STEROL REGULATORY ELEMENT BINDING PROTEIN (SREBP) CHAPERONE (SCAP) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The invention relates to double stranded ribonucleic acid (dsRNAi) agents and compositions targeting the SCAP gene, as well as methods of inhibiting expression of a SCAP gene and methods of treating subjects having a SCAP-associated disorder, such as nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), using such dsRNAi agents and compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/002,034, filed on Jun. 7, 2018, which is a 35 § U.S.C. 111(a)continuation application which claims the benefit of priority toPCT/US2016/065781, filed on Dec. 9, 2016, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 62/265,580, filed onDec. 10, 2015, and to U.S. Provisional Patent Application No.62/378,964, filed on Aug. 24, 2016. The entire contents of each of theforegoing patent applications are hereby incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jun. 30, 2020, is named121301_05004_SL.txt and is 606,736 bytes in size.

BACKGROUND OF THE INVENTION

The accumulation of excess triglyceride in the liver is known as hepaticsteatosis (or fatty liver), and is associated with adverse metabolicconsequences, including insulin resistance and dyslipidemia. Fatty liveris frequently found in subjects having excessive alcohol intake andsubjects having obesity, diabetes, or hyperlipidemia. However, in theabsence of excessive alcohol intake (>10 g/day), nonalcoholic fattyliver disease (NAFLD) can develop. NAFLD refers to a wide spectrum ofliver diseases that can progress from simple fatty liver (steatosis), tononalcoholic steatohepatitis (NASH), to cirrhosis (irreversible,advanced scarring of the liver). All of the stages of NAFLD have incommon the accumulation of fat (fatty infiltration) in the liver cells(hepatocytes).

The NAFLD spectrum begins with and progress from its simplest stage,called simple fatty liver (steatosis). Simple fatty liver involves theaccumulation of fat (triglyceride) in the liver cells with noinflammation (hepatitis) or scarring (fibrosis). The next stage anddegree of severity in the NAFLD spectrum is NASH, which involves theaccumulation of fat in the liver cells, as well as inflammation of theliver. The inflammatory cells destroy liver cells (hepatocellularnecrosis), and NASH ultimately leads to scarring of the liver(fibrosis), followed by irreversible, advanced scarring (cirrhosis).Cirrhosis that is caused by NASH is the last and most severe stage inthe NAFLD spectrum.

NAFLD is now the leading cause of chronic liver disease in the UnitedStates and there are currently about 1 million patients diagnosed withNASH. Subjects having NASH have a doubled risk of cardiovascularmortality as compared to the general population and NASH is the thirdmost common casue of liver transplantation.

A central element in the regulation of lipid biosynthesis in the humanliver is a group of transcription factors termed Sterol RegulatoryElement Binding Proteins (SREBPs). There are three SREBP isoforms calledSREBP-1a, SREBP-1c and SREBP-2. They are located in the endoplasmaticreticulum (ER) in precursor form (Yokoyama C. et al., Cell 1993, 75:187;Hua X. et al., Proc. Natl. Acad. Sci. 1993, 90:11603) which, in thepresence of cholesterol, is bound to cholesterol and two other proteins:SCAP (SREBF chaperone) and Insig1 (Insulin-induced gene 1). Whencholesterol levels fall, Insig-1 dissociates from the SREBP-SCAPcomplex, allowing the complex to migrate to the Golgi apparatus, whereSREBP is cleaved by S1P and S2P (site 1/2 protease; Sakai J et al, Mol.Cell. 1998, 2:505; Rawson R. B. et al, Mol. Cell. 1997, 1:47), twoenzymes that are activated by SCAP. The cleaved SREBP then migrates tothe nucleus and acts as a transcription factor by binding to the SRE(sterol regulatory element) of a number of genes and stimulating theirtranscription (Briggs M. R. et al., J. Biol. Chem. 1993, 268:14490).Among the genes transcribed are the LDL-Receptor, up-regulation of whichleads to increased in-flux of cholesterol from the bloodstream, HMG-CoAreductase, the rate limiting enzyme in de-novo cholesterol synthesis(Anderson et al, Trends Cell Biol 2003, 13:534), as well as a number ofgenes involved in fatty acid synthesis, e.g., Patatin-Like PhospholipaseDomain Containing 3 (PNPLA3), which is a multifunctional enzyme whichhas both triacylglycerol lipase (triglyceride hydrolysis) andacylglycerol O-acyltransferase activities (triglyceride synthesis).

Since SCAP-binding is a prerequisite for the transport and activation ofall three SREBP isoforms, inhibition of the expression and/or activityof SCAP with an agent that can selectively and efficiently attenuate thebody's own lipid biosynthesis, e.g. by inhibiting SCAP, and therebySREBP activity, would be useful for treating pathological processesmediated directly or indirectly by SCAP expression.

Currently, treatments for NAFLD are directed towards weight loss andtreatment of any secondary conditions, such as insulin resistance ordyslipidemia. However, to date, no pharmacologic treatments for NAFLDhave been approved. Therefore, there is a need for therapies forsubjects suffering from NAFLD, e.g., steatosis and NASH.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a sterol regulatory element binding protein (SREBP)chaperone (SCAP) gene. The SCAP gene may be within a cell, e.g., a cellwithin a subject, such as a human. The present invention also providesmethods of using the iRNA compositions of the invention for inhibitingthe expression of a SCAP gene and/or for treating a subject who wouldbenefit from inhibiting or reducing the expression of a SCAP gene, e.g.,a subject suffering or prone to suffering from a SCAP-associateddisease, for example, Nonalcoholic Fatty Liver Disease (NAFLD), e.g.,nonalcoholic steatohepatitis (NASH).

Accordingly, in one aspect, the present invention provides doublestranded ribonucleic acids (RNAi) agents for inhibiting expression of asterol regulatory element binding protein (SREBP) chaperone (SCAP) gene.The double stranded RNAi agents include a sense strand and an antisensestrand, wherein the sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NOs:1-13, wherein the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ IDNOs:14-26.

In another aspect, the present invention provides a double strandedribonucleic acid (RNAi) agent for inhibiting expression of a SCAP gene,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand, wherein the antisense strand comprising a region ofcomplementarity which comprises at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the antisensesequences listed in any one of Tables 2, 3, 5 and 6.

In some embodiments, the sense and antisense strands of the doublestranded RNAi agent of the present invention comprise sequences selectedfrom the group consisting of any one of the sequences in any one ofTables 2, 3, 5 and 6.

In some aspects, the sense strand comprises at least 15 contiguousnucleotides differnting by no more than 3 nucleotides from thenucleotide sequence of nucleotides 345-363, 378-396, 383-401, 402-420,437-455, 458-476, 491-509, 1014-1032, 1237-1255, 1243-1261, 1259-1277,1318-1336, 1323-1341, 1497-1515, 1571-1589, 1588-1606, 1605-1623,1725-1743, 1767-1785, 1946-1964, 2004-2022, 2160-2178, 2193-2211,2217-2235, 2517-2535, 2547-2565, 2616-2634, 2663-2681, 2717-2735,2734-2752, 2751-2769, 2874-2892, 2885-2903, 2998-3016, 3276-3294,3292-3310, 3325-3343, 3342-3360, 3420-3438, 3469-3487, 3478-3496,3533-3551, 3549-3567, 3565-3583, 3579-3597, 3622-3640, 3667-3685,3771-3789, 3788-3806, 3871-3889, 3891-3909, 3904-3922, 3921-3939,4002-4020, 4010-4028, 4027-4045, 4075-4093, 4129-4147, 4145-4163,4149-4167, 4168-4186, 4184-4202 and 4197-4215 of SEQ ID NO: 2.

In some embodiments, the antisense strand comprises at least 15contiguous nucleotides differting by no more than 3 nucleotides from theantisense nucleotide sequence of a duplex selected from the groupconsisting of AD-77633, AD-77631, AD-77630, AD-77629, AD-77627,AD-77625, AD-77624, AD-77587, AD-77573, AD-77572, AD-77571, AD-77567,AD-77566, AD-77738, AD-77731, AD-77730, AD-77729, AD-77721, AD-77718,AD-77706, AD-77701, AD-77690, AD-77688, AD-77686, AD-77669, AD-77667,AD-77662, AD-77660, AD-77656, AD-77655, AD-77654, AD-77555, AD-77554,AD-77547, AD-77530, AD-77529, AD-77527, AD-77526, AD-77521, AD-77519,AD-77518, AD-77514, AD-77513, AD-77512, AD-77510, AD-77507, AD-77505,AD-77499, AD-77498, AD-77494, AD-77492, AD-77491, AD-77490, AD-77486,AD-77485, AD-77484, AD-77483, AD-77479, AD-77478, AD-77477, AD-77476,AD-77475 and AD-77474 as listed in Tables 5 and 6.

In certain embodiments, the double stranded RNAi agent comprises atleast one modified nucleotide. In certain embodiments, the doublestranded RNAi agent comprises no more than 4 (i.e., 4, 3, 2, 1, or 0)unmodified nucleotides in the sense strand. In certain embodiments, thedouble stranded RNAi agent comprises no more than 4 (i.e., 4, 3, 2, 1,or 0) unmodified nucleotides in the antisense strand. In certainembodiments, the dsRNA agent comprises no more than 4 (i.e., 4, 3, 2, 1,or 0) unmodified nucleotides in both the sense strand and the antisensestrand. In certain embodiments, substantially all of the nucleotides inthe sense strand of the double stranded RNAi agent are modifiednucleotides. In certain embodiments, substantially all of thenucleotides in the antisense strand of the double stranded RNAi agentare modified nucleotides. In certain embodiments, all of the nucleotidesin the sense strand of the double stranded RNAi agent and all of thenucleotides of the antisense strand are modified nucleotides.

In certain embodiments, the modified nucleotide(s) is/are independentlyselected from the group consisting of a 2′-O-methyl modified nucleotide,a nucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative or a dodecanoic acidbisdecylamide group.

In one embodiment, the modified nucleotide is selected from the groupconsisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, a nucleotide comprising a5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotidecomprising adenosine-glycol nucleic acid (GNA), a nucleotide comprisingthymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising2′-deoxythymidine-3′ phosphate, a nucleotide comprising2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to acholesteryl derivative and a dodecanoic acid bisdecylamide group

In certain embodiments, the modified nucleotide is selected from thegroup consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, and a non-natural basecomprising nucleotide. In some embodiments, the modified nucleotidescomprise a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).In certain embodiments, the modifications on the nucleotides are2′-O-methyl and 2′ fluoro modifications.

In some embodiments, the double stranded RNAi agent further comprises atleast one phosphorothioate internucleotide linkage. In some embodiments,the double stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages, e.g., 6, 7, or 8 phosphorothioateinternucleotide linkages.

In certain embodiments, the double stranded RNAi agent comprises aregion of complementarity at least 17 nucleotides in length. In certainembodiments, the double stranded RNAi agent comprises a region ofcomplementarity 19 and 23 nucleotides in length. In certain embodiments,the double stranded RNAi agent comprises a region of complementarity is19 nucleotides in length.

In certain embodiments, each strand of the double stranded RNAi agent isno more than 30 nucleotides in length. In certain embodiments, thedouble stranded RNAi agent is at least 15 nucleotides in length.

In certain embodiments, at least one strand of the double stranded RNAiagent comprises a 3′ overhang of at least 1 nucleotide. In certainembodiments, the at least one strand comprises a 3′ overhang of at least2 nucleotides.

In some embodiment, the double stranded RNAi agent further comprises aligand. In one embodiment, the ligand is conjugated to the 3′ end of thesense strand of the double stranded RNAi agent. In one embodiment, theligand is conjugated to the 3′ end of the sense strand through amonovalent or a branched bivalent or trivalent linker. In oneembodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivativeconjugated to the 3′ end of the sense strand of the double stranded RNAiagent. In certain embodiments, the ligand is conjugated to the 3′ end ofthe sense strand of the double stranded RNAi agent through a branchedbivalent or trivalent linker. In certain embodiments, the ligand is

In certain embodiments, the double stranded RNAi agent is conjugated tothe ligand as shown in the following schematic

and, wherein X is O or S.

In some embodiments, the X is O. In certain embodiments, the ligand is acholesterol.

In some embodiments, the region of complementarity comprises any one ofthe antisense sequences in any one of Table 2, Table 3, Table 5 andTable 6. In other embodiments, the region of complementarity consists ofany one of the antisense sequences in any one of Table 2, Table 3, Table5 and Table 6.

In one aspect, the invention provides a double stranded ribonucleic acid(RNAi) agent for inhibiting expression of a sterol regulatory elementbinding protein (SREBP) chaperone (SCAP) gene, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present, independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;

modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; and whereinthe sense strand is conjugated to at least one ligand.

In some embodiments, i is 0; j is 0; i is 1; j is 1; both i and j are 0;or both i and j are 1. In some embodiments, k is 0; l is 0; k is 1; l is1; both k and l are 0; or both k and l are 1.

In some embodiments, XXX is complementary to X′X′X′, YYY iscomplementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′. In someembodiments, the YYY motif occurs at or near the cleavage site of thesense strand. In some embodiments, the Y′Y′Y′ motif occurs at the 11, 12and 13 positions of the antisense strand from the 5′-end. In someembodiments, the Y′ is 2′-O-methyl.

In some embodiments, formula (III) is represented by formula (IIIa):

sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIa).

In some embodiments, formula (III) is represented by formula (IIIb):

sense: 5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′ 5′ (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In some embodiments, formula (III) is represented by formula (IIIc):

sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides.

In some embodiments, formula (III) is represented by formula (IIId):

sense: 5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′antisense: 3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′ 5′ (IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.

In some embodiments, the double stranded region is 15-30 nucleotidepairs in length. In some embodiments, the double stranded region is17-23 nucleotide pairs in length. In some embodiments, the doublestranded region is 17-25 nucleotide pairs in length. In someembodiments, the double stranded region is 23-27 nucleotide pairs inlength. In some embodiments, the double stranded region is 19-21nucleotide pairs in length. In some embodiments, the double strandedregion is 21-23 nucleotide pairs in length. In some embodiments, eachstrand has 15-30 nucleotides. In some embodiments, each strand has 19-30nucleotides.

In some embodiments, the modifications on the nucleotides are selectedfrom the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl,and combinations thereof. In some embodiments, the modifications on thenucleotides are 2′-O-methyl or 2′-fluoro modifications.

In some embodiments, the ligand is one or more GalNAc derivativesattached through a bivalent or trivalent branched linker; or acholesterol.

In some embodiments, the ligand is

In some embodiments, the ligand is attached to the 3′ end of the sensestrand. In some embodiments, the RNAi agent is conjugated to the ligandas shown in the following schematic

In some embodiments, the agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage. In someembodiments, the phosphorothioate or methylphosphonate internucleotidelinkage is at the 3′-terminus of one strand. In some embodiments, thestrand is the antisense strand. In some embodiments, the strand is thesense strand.

In some embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand. In someembodiments, strand is the antisense strand. In some embodiments, strandis the sense strand.

In some embodiments, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In some embodiments, strand is the antisense strand.

In some embodiments, the base pair at the 1 position of the 5′-end ofthe antisense strand of the duplex is an AU base pair. In someembodiments, the Y nucleotides contain a 2′-fluoro modification. In someembodiments, the Y′ nucleotides contain a 2′-O-methyl modification. Insome embodiments, p′>0. In some embodiments, p′=2. In some embodiments,q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to thetarget mRNA. In some embodiments, q′=0, p=0, q=0, and p′ overhangnucleotides are non-complementary to the target mRNA.

In some embodiments, the sense strand has a total of 21 nucleotides andthe antisense strand has a total of 23 nucleotides. In some embodiments,at least one n_(p)′ is linked to a neighboring nucleotide via aphosphorothioate linkage. In some embodiments, all n_(p)′ are linked toneighboring nucleotides via phosphorothioate linkages.

In some embodiments, the RNAi agent is selected from the group of RNAiagents listed in any one of Table 2, Table 3, Table 5 and Table 6. Insome embodiments, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In another aspect, the invention provides cells containing the doublestranded RNAi agents provided herein.

In yet another aspect, the invention provides pharmaceuticalcompositions for inhibiting expression of a SCAP gene. The compositionsinclude the double stranded RNAi agents provided herein. In certainembodiments, the RNAi agent is administered in an unbuffered solution.In some embodiments, the unbuffered solution is saline or water. Inother embodiments, the RNAi agent is administered with a buffersolution. In some embodiments, the buffer solution comprises acetate,citrate, prolamine, carbonate, or phosphate or any combination thereof.In some embodiments, the buffer solution is phosphate buffered saline(PBS). In some embodiments, the pharmaceutical compositions furthercomprise a lipid formulation. In some embodiments, the lipid formulationcomprises a LNP. In some embodiments, the lipid formulation comprises aMC3.

In one aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; modifications on N_(b) differ from the modification on Yand modifications on N_(b)′ differ from the modification on Y; andwherein the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; and whereinthe sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; and whereinthe sense strand is conjugated to at least one ligand, wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.

In yet another aspect, the present invention provides a double strandedRNAi agent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; herein thesense strand comprises at least one phosphorothioate linkage; andwherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (III)wherein: each n_(p), n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide; p, q, and q′are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via a phosphorothioate linkage; each N_(a)and N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 nucleotides which are either modified or unmodified orcombinations thereof, each sequence comprising at least two differentlymodified nucleotides; YYY and Y′Y′Y′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; wherein the sense strand comprises at least onephosphorothioate linkage; and wherein the sense strand is conjugated toat least one ligand, wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker.

In one aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; p, p′, q, and q′are each independently 0-6; each N_(a) and N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 0-25 nucleotides whichare either modified or unmodified or combinations thereof, each sequencecomprising at least two differently modified nucleotides; each N_(b) andN_(b)′ independently represents an oligonucleotide sequence comprising0-10 nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; modifications on N_(b) differ from the modification on Yand modifications on N_(b)′ differ from the modification on Y; andwherein the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; and whereinthe sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe sense strand is conjugated to at least one ligand, wherein theligand is one or more GalNAc derivatives attached through a bivalent ortrivalent branched linker.

In yet another aspect, the present invention provides a double strandedRNAi agent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: i, j, k, and l are each independently 0 or 1; each n_(p),n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide; p, q, and q′ are eachindependently 0-6; n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via a phosphorothioate linkage; each N_(a) andN_(a)′ independently represents an oligonucleotide sequence comprising0-25 nucleotides which are either modified or unmodified or combinationsthereof, each sequence comprising at least two differently modifiednucleotides; each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-10 nucleotides which are eithermodified or unmodified or combinations thereof; XXX, YYY, ZZZ, X′X′X′,Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of threeidentical modifications on three consecutive nucleotides, and whereinthe modifications are 2′-O-methyl or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; herein thesense strand comprises at least one phosphorothioate linkage; andwherein the sense strand is conjugated to at least one ligand, whereinthe ligand is one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In another aspect, the present invention provides a double stranded RNAiagent inhibiting expression of a sterol regulatory element bindingprotein (SREBP) chaperone (SCAP) gene in a cell, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to part of an mRNA encoding SCAP, wherein each strand isabout 14 to about 30 nucleotides in length, wherein the double strandedRNAi agent is represented by formula (III):

sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIa)wherein: each n_(p), n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide; p, q, and q′are each independently 0-6; n_(p)′>0 and at least one n_(p)′ is linkedto a neighboring nucleotide via a phosphorothioate linkage; each N_(a)and N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 nucleotides which are either modified or unmodified orcombinations thereof, each sequence comprising at least two differentlymodified nucleotides; YYY and Y′Y′Y′ each independently represent onemotif of three identical modifications on three consecutive nucleotides,and wherein the modifications are 2′-O-methyl or 2′-fluoromodifications; wherein the sense strand comprises at least onephosphorothioate linkage; and wherein the sense strand is conjugated toat least one ligand, wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker.

In yet another aspect, the present invention provides a double strandedribonucleic acid (RNAi) agent for inhibiting expression of a sterolregulatory element binding protein (SREBP) chaperone (SCAP) gene,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the nucleotide sequences of SEQ IDNOs:1-13 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NOs:14-26, wherein substantially all ofthe nucleotides of the sense strand comprise a modification selectedfrom the group consisting of a 2′-O-methyl modification and a 2′-fluoromodification, wherein the sense strand comprises two phosphorothioateinternucleotide linkages at the 5′-terminus, wherein substantially allof the nucleotides of the antisense strand comprise a modificationselected from the group consisting of a 2′-O-methyl modification and a2′-fluoro modification, wherein the antisense strand comprises twophosphorothioate internucleotide linkages at the 5′-terminus and twophosphorothioate internucleotide linkages at the 3′-terminus, andwherein the sense strand is conjugated to one or more GalNAc derivativesattached through a branched bivalent or trivalent linker at the3′-terminus.

In another aspect, the present invention provides a double strandedribonucleic acid (RNAi) agent for inhibiting expression of a sterolregulatory element binding protein (SREBP) chaperone (SCAP) gene,wherein the double stranded RNAi agent comprises a sense strand and anantisense strand forming a double stranded region, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the nucleotide sequences of SEQ IDNOs:1-13 and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NOs:14-26, wherein the sense strandcomprises at least one 3′-terminal deoxy-thymine nucleotide (dT), andwherein the antisense strand comprises at least one 3′-terminaldeoxy-thymine nucleotide (dT).

In another aspect, the invention provides cells containing the doublestranded RNAi agents provided herein.

The invention also provides methods of inhibiting SCAP expression in acell. The methods include contacting the cell with the double strandedRNAi agent or the pharmaceutical composition as described herein; andmaintaining the cell for a time sufficient to obtain degradation of themRNA transcript of a SCAP gene, thereby inhibiting expression of theSCAP gene in the cell.

In certain embodiments, the cell is within a subject. In certainembodiments, the subject is a human. In one embodiment, the subject is afemale human. In another embodiment, the subject is a male human.

In certain embodiments, the human subject suffers from a SCAP-associateddisease. In one embodiment, the SCAP-associated disease is nonalcoholicfatty liver disease (NAFLD). In another embodiment, the SCAP-associateddisease is fatty liver (steatosis). In another embodiment, theSCAP-associated disease is nonalcoholic steatohepatitis (NASH). In yetanother embodiment, the SCAP-associated disease is obesity. In yetanother embodiment, the SCAP-associated disease is insulin resistance.

In certain embodiments, the SCAP expression is inhibited by at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85% or at leastabout 90%.

In another aspect, the invention provides methods of treating a subjecthaving a disorder that would benefit from reduction in SCAP expression.The methods include administering to the subject a therapeuticallyeffective amount of any of the double stranded RNAi agents or thepharmaceutical composition provided herein, thereby treating thesubject.

In certain embodiments, the human subject suffers from a SCAP-associateddisease.

In one embodiment, the SCAP-associated disease is nonalcoholic fattyliver disease (NAFLD). In another embodiment, the SCAP-associateddisease is fatty liver (steatosis). In another embodiment, theSCAP-associated disease is nonalcoholic steatohepatitis (NASH). In yetanother embodiment, the SCAP-associated disease is obesity. In yetanother embodiment, the SCAP-associated disease is insulin resistance.

In certain embodiments, administration of the double stranded RNAi agentto the subject causes a decrease in one or more serum lipid, e.g.,triglycerides, and/or a decrease in SCAP protein accumulation. Incertain embodiments, the administration of the double stranded RNAiagent to the subject causes a decrease in PNPLA3 protein accumulation orSREBP protein accumulation.

In certain embodiments, the method of the present invention furthercomprises administering an additional therapeutic agent and/ortreatment, e.g., life-style changes, e.g., exercise, weight loss, dietchange, antioxidant therapy, intake of omega-3-fatty acids, and/or livertransplant.

In some embodiments, the double stranded RNAi agent is administered at adose of about 0.01 mg/kg to about 50 mg/kg. In other embodiments, thedouble stranded RNAi agent is administered to the subjectsubcutaneously.

In a further aspect, the present invention also provides methods ofinhibiting the expression of SCAP in a subject. The methods includeadministering to the subject a therapeutically effective amount of anyof the double stranded RNAi agents or the pharmaceutical compositionprovided herein, thereby inhibiting the expression of SCAP in thesubject.

In another aspect, the present invention provides methods of decreasingthe plasma triglyceride level in a subject. The methods includeadministering to the subject a therapeutically effective amount of anyof the double stranded RNAi agents or the pharmaceutical compositionprovided herein, thereby decreasing the plasma triglyceride level in thesubject.

In another aspect, the present invention provides methods of inhibitingthe progression of NAFLD in a subject, such as the progression ofsteatosis to NASH in a subject having steatosis or a subject at risk ofdeveloping steatosis, e.g., a subject having insulin resistance or anobese subject; or the progression of NASH to cirrhosis in a subjecthaving NASH or a subject at risk of developing cirrhosis. The methodsinclude administering to the subject a therapeutically effective amountof any of the double stranded RNAi agents or the pharmaceuticalcomposition provided herein, thereby inhibiting the progression of NAFLDin the subject.

In some embodiments, the methods of the invention may further includedetermining serum aspartate aminotransferase (AST) levels in thesubject, alanine aminotransferase (ALT) levels in the subject, plasmatriglyceride levels in the subject and/or determining the liver fatlevel in the subject In yet another aspect, the invention provides kitsfor performing the methods of the invention. In one embodiment, theinvention provides a kit for performing a method of inhibitingexpression of SCAP gene in a cell by contacting a cell with a doublestranded RNAi agent of the invention in an amount effective to inhibitexpression of the SCAP in the cell. The kit comprises an RNAi agent andinstructions for use and, optionally, means for administering the RNAiagent to a subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a sterol regulatory element binding protein (SREBP)chaperone (SCAP) gene. The SCAP gene may be within a cell, e.g., a cellwithin a subject, such as a human. The present invention also providesmethods of using the iRNA compositions of the invention for inhibitingthe expression of a SCAP gene and/or for treating a subject having adisorder that would benefit from inhibiting or reducing the expressionof a SCAP gene, e.g., a SCAP-associated disease, for example,Nonalcoholic Fatty Liver Disease (NAFLD), e.g., nonalcoholicsteatohepatitis (NASH).

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is about 30 nucleotides or less in length, e.g.,15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which regionis substantially complementary to at least part of an mRNA transcript ofa SCAP gene.

In certain embodiments, the iRNAs of the invention include an RNA strand(the antisense strand) which can include longer lengths, for example upto 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53nucleotides in length with a region of at least 19 contiguousnucleotides that is substantially complementary to at least a part of anmRNA transcript of a C3 gene. These iRNAs with the longer lengthantisense strands preferably include a second RNA strand (the sensestrand) of 20-60 nucleotides in length wherein the sense and antisensestrands form a duplex of 18-30 contiguous nucleotides.

The use of these iRNAs enables the targeted degradation of mRNAs of aSCAP gene in mammals. Very low dosages of SCAP iRNAs, in particular, canspecifically and efficiently mediate RNA interference (RNAi), resultingin significant inhibition of expression of a SCAP gene. Using cell-basedassays, the present inventors have demonstrated that iRNAs targetingSCAP can mediate RNAi, resulting in significant inhibition of expressionof a SCAP gene. Thus, methods and compositions including these iRNAs areuseful for treating a subject who would benefit by a reduction in thelevels and/or activity of a SCAP protein, such as a subject having aSCAP-associated disease, for example, Nonalcoholic Fatty Liver Disease(NAFLD), e.g., nonalcoholic steatohepatitis (NASH).

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of a SCAP gene,as well as compositions and methods for treating subjects havingdiseases and disorders that would benefit from inhibition and/orreduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”. The term “or” is usedherein to mean, and is used interchangeably with, the term “and/or,”unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, aboutmeans±10%. In certain embodiments, about means±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understoodto include the number adjacent to the term “at least”, and allsubsequent numbers or integers that could logically be included, asclear from context. For example, the number of nucleotides in a nucleicacid molecule must be an integer. For example, “at least 18 nucleotidesof a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21nucleotides have the indicated property. When at least is present beforea series of numbers or a range, it is understood that “at least” canmodify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the valueadjacent to the phrase and logical lower values or intergers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range.

The term “SCAP” refers to sterol regulatory element binding protein(SREBP) chaperone, also known as SREBP cleavage activating protein,having an amino acid sequence from any vertebrate or mammalian source,including, but not limited to, human, bovine, chicken, rodent, mouse,rat, porcine, ovine, primate, monkey, and guinea pig, unless specifiedotherwise. The term also refers to fragments and variants of native SCAPthat maintain at least one in vivo or in vitro activity of a nativeSCAP. The term encompasses full-length unprocessed precursor forms ofSCAP as well as mature forms resulting from post-translational cleavageof the signal peptide. The nucleotide and amino acid sequence of a humaSCAP can be found at, for example, GenBank Accession No. GI: 66932901(NM_012235.2; SEQ ID NO:1).

The nucleotide and amino acid sequence of a huma SCAP may also be foundat, for example, GenBank Accession No. GI: 987996597 (NM_012235.3; SEQID NO:2); GenBank Accession No. GI: 530372097 (XM_005264967.1; SEQ IDNO:3); GenBank Accession No. GI: 530372099 (XM_005264968.1; SEQ IDNO:4); GenBank Accession No. GI: 530372101 (XM_005264969.1; SEQ IDNO:5); GenBank Accession No. GI: 767922691 (XM_011533501.1; SEQ IDNO:6); GenBank Accession No. GI: 767922693 (XM_011533502.1; SEQ IDNO:7); GenBank Accession No. GI: 767922696 (XM_005264970.3; SEQ IDNO:8); GenBank Accession No. GI: 530372105 (XM_005264971.1; SEQ IDNO:9); and GenBank Accession No. GI: 767922697 (XM_005264972.3; SEQ IDNO:10).

The nucleotide and amino acid sequence of a Cynomolgus monkey SCAP canbe found at, for example, GenBank Accession No. GI: 544413972(XM_005546963.1; SEQ ID NO:11). The nucleotide and amino acid sequenceof a mouse SCAP can be found at, for example, GenBank Accession No. GI:557636668 (NM_001103162.2; SEQ ID NO:12). The nucleotide and amino acidsequence of a rat SCAP can be found at, for example, GenBank AccessionNo. GI: 155369286 (NM_001100966.1; SEQ ID NO:13). Additional examples ofSCAP sequences are readily available using publicly available databases,e.g., GenBank, UniProt, and OMIM.

The term “SCAP” as used herein also refers to a particular polypeptideexpressed in a cell by naturally occurring DNA sequence variations ofthe SCAP gene, such as a single nucleotide polymorphism in the SCAPgene. Numerous SNPs within the SCAP gene have been identified and may befound at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp).Non-limiting examples of SNPs within the SCAP gene may be found at, NCBIdbSNP Accession Nos. rs926103; rs12487736; rs12490383; rs754498;rs877097; rs878659; rs881264; rs900690; rs900691; rs900692; rs909200;rs909201; rs928391; or rs943555.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a SCAP gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of a SCAPgene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 1). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of SCAP in a cell, e.g., a cell within a subject, such asa mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNAi that interacts with a target RNA sequence, e.g., an SCAPtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into double-stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processesthese dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the invention relates to a singlestranded RNA (ssRNA) (the antisense strand of an siRNA duplex) generatedwithin a cell and which promotes the formation of a RISC complex toeffect silencing of the target gene, i.e., an SCAP gene. Accordingly,the term “siRNA” is also used herein to refer to an RNAi as describedabove.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No. 8,101,348and in Lima et al., (2012) Cell 150:883-894, the entire contents of eachof which are hereby incorporated herein by reference. Any of theantisense nucleotide sequences described herein may be used as asingle-stranded siRNA as described herein or as chemically modified bythe methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions and methodsof the invention is a double stranded RNA and is referred to herein as a“double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,”“dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex ofribonucleic acid molecules, having a duplex structure comprising twoanti-parallel and substantially complementary nucleic acid strands,referred to as having “sense” and “antisense” orientations with respectto a target RNA, i.e., a SCAP gene. In some embodiments of theinvention, a double stranded RNA (dsRNA) triggers the degradation of atarget RNA, e.g., an mRNA, through a post-transcriptional gene-silencingmechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/or amodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides. Insome embodiments, the hairpin loop can be 10 or fewer nucleotides. Insome embodiments, the hairpin loop can be 8 or fewer unpairednucleotides. In some embodiments, the hairpin loop can be 4-10 unpairednucleotides. In some embodiments, the hairpin loop can be 4-8nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs. In one embodiment of theRNAi agent, at least one strand comprises a 3′ overhang of at least 1nucleotide. In another embodiment, at least one strand comprises a 3′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In other embodiments, at least one strandof the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. Incertain embodiments, at least one strand comprises a 5′ overhang of atleast 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or15 nucleotides. In still other embodiments, both the 3′ and the 5′ endof one strand of the RNAi agent comprise an overhang of at least 1nucleotide.

In one embodiment, an RNAi agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., a SCAP target mRNA sequence, to direct thecleavage of the target RNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end.In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide,e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the3′-end and/or the 5′-end. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or theantisense strand, or both, can include extended lengths longer than 10nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30nucleotides, or 10-15 nucleotides in length. In certain embodiments, anextended overhang is on the sense strand of the duplex. In certainembodiments, an extended overhang is present on the 3′ end of the sensestrand of the duplex. In certain embodiments, an extended overhang ispresent on the 5′ end of the sense strand of the duplex. In certainembodiments, an extended overhang is on the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the3′ end of the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 5′ end of the antisense strand ofthe duplex. In certain embodiments, one or more of the nucleotides inthe overhang is replaced with a nucleoside thiophosphate. In certainembodiments, the overhang includes a self-complementary portion suchthat the overhang is capable of forming a hairpin structure that isstable under physiological conditions.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will be doublestranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a SCAP mRNA.

As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example a target sequence, e.g., a SCAP nucleotidesequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding SCAP). For example, a polynucleotide iscomplementary to at least a part of a SCAP mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding SCAP.

Accordingly, in some embodiments, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target SCAP sequence. Inother embodiments, the antisense strand polynucleotides disclosed hereinare substantially complementary to the target SCAP sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of SEQ ID NOs:1-13, or a fragment of SEQ IDNOs:1-13, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target SCAP sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to any one of the sense strand nucleotidesequences in any one of Tables 2, 3, 5, or 6, or a fragment of any oneof the sense strand nucleotide sequences in any one of Tables 2, 3, 5,or 6, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is the same as a target SCAP sequence,and wherein the sense strand polynucleotide comprises a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of SEQID NOs:14-26, or a fragment of any one of SEQ ID NOs:14-26, such asabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about% 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% complementary.

In one embodiment, at least partial suppression of the expression of aSCAP gene, is assessed by a reduction of the amount of SCAP mRNA whichcan be isolated from or detected in a first cell or group of cells inwhich a SCAP gene is transcribed and which has or have been treated suchthat the expression of a SCAP gene is inhibited, as compared to a secondcell or group of cells substantially identical to the first cell orgroup of cells but which has or have not been so treated (controlcells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing”or “delivering the iRNA into the cell” by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an iRNA canoccur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an iRNA into a cell may be invitro and/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vitrointroduction into a cell includes methods known in the art such aselectroporation and lipofection. Further approaches are described hereinbelow and/or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in SCAP expression; a human at risk for adisease, disorder or condition that would benefit from reduction in SCAPexpression; a human having a disease, disorder or condition that wouldbenefit from reduction in SCAP expression; and/or human being treatedfor a disease, disorder or condition that would benefit from reductionin SCAP expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with SCAP geneexpression and/or SCAP protein production, e.g., fatty liver(steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of theliver, accumulation of fat in the liver, inflammation of the liver,hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fattyliver disease (NAFLD). “Treatment” can also mean prolonging survival ascompared to expected survival in the absence of treatment.

The term “lower” in the context of the level of SCAP in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more. In certain embodiments, a decrease is atleast 20%. “Lower” in the context of the level of SCAP in a subject ispreferably down to a level accepted as within the range of normal for anindividual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of a SCAP gene and/or production of SCAPprotein, refers to a reduction in the likelihood that a subject willdevelop a symptom associated with such a disease, disorder, orcondition, e.g., a symptom of SCAP gene expression, such as the presenceof fatty liver (steatosis), nonalcoholic steatohepatitis (NASH),cirrhosis of the liver, accumulation of fat in the liver, inflammationof the liver, hepatocellular necrosis, liver fibrosis, obesity, ornonalcoholic fatty liver disease (NAFLD). The failure to develop adisease, disorder or condition, or the reduction in the development of asymptom associated with such a disease, disorder or condition (e.g., byat least about 10% on a clinically accepted scale for that disease ordisorder), or the exhibition of delayed symptoms delayed (e.g., by days,weeks, months or years) is considered effective prevention.

As used herein, the term “SCAP-associated disease,” is a disease ordisorder that is caused by, or associated with SCAP gene expression orSCAP protein production. The term “SCAP-associated disease” includes adisease, disorder or condition that would benefit from a decrease inSCAP gene expression, replication, or protein activity. Non-limitingexamples of SCAP-associated diseases include, for example, fatty liver(steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of theliver, accumulation of fat in the liver, inflammation of the liver,hepatocellular necrosis, liver fibrosis, obesity, or nonalcoholic fattyliver disease (NAFLD), hyperlipidemia, hyperlipoproteinemia,hypercholesterolemis, hypertriglyceridemia, atherosclerosis,pancreatitis, non-insulin dependent diatebes mellitus, coronary heartdisease, and cerebrovascular disease. In one embodiment, theSCAP-associated disease is nonalcoholic fatty liver disease (NAFLD). Inanother embodiment, the SCAP-associated disease is nonalcoholicsteatohepatitis (NASH). In another embodiment, the SCAP-associateddisease is liver cirrhosis. In another embodiment, the SCAP-associateddisease is insulin resistance. In another embodiment, theSCAP-associated disease is not insulin resistance. In one embodiment,the SCAP-associated disease is obesity.

In one embodiment, a SCAP-associated disease is nonalcoholic fatty liverdisease (NAFLD). As used herein, “nonalcoholic fatty liver disease,”used interchangeably with the term “NAFLD,” refers to a disease definedby the presence of macrovascular steatosis in the presence of less than20 gm of alcohol ingestion per day. NAFLD is the most common liverdisease in the United States, and is commonly associated with insulinresistance/type 2 diabetes mellitus and obesity. NAFLD is manifested bysteatosis, steatohepatitis, cirrhosis, and sometimes hepatocellaularcarcinoma. For a review of NAFLD, see Tolman and Dalpiaz (2007) Ther.Clin. Risk. Manag., 3(6):1153-1163 the entire contents of which areincorporated herein by reference.

As used herein, the terms “steatosis,” “hepatic steatosis,” and “fattyliver disease” refer to the accumulation of triglycerides and other fatsin the liver cells.

As used herein, the term “Nonalcoholic steatohepatitis” or “NASH” refersto liver inflammation and damage caused by a buildup of fat in theliver. NASH is part of a group of conditions called nonalcoholic fattyliver disease (NAFLD). NASH resembles alcoholic liver disease, butoccurs in people who drink little or no alcohol. The major feature inNASH is fat in the liver, along with inflammation and damage. Mostpeople with NASH feel well and are not aware that they have a liverproblem. Nevertheless, NASH can be severe and can lead to cirrhosis, inwhich the liver is permanently damaged and scarred and no longer able towork properly. NASH is usually first suspected in a person who is foundto have elevations in liver tests that are included in routine bloodtest panels, such as alanine aminotransferase (ALT) or aspartateaminotransferase (AST). When further evaluation shows no apparent reasonfor liver disease (such as medications, viral hepatitis, or excessiveuse of alcohol) and when x rays or imaging studies of the liver showfat, NASH is suspected. The only means of proving a diagnosis of NASHand separating it from simple fatty liver is a liver biopsy.

As used herein, the term “cirrhosis,” defined histologically, is adiffuse hepatic process characterized by fibrosis and conversion of thenormal liver architecture into structurally abnormal nodules.

As used herein, the term “serum lipid” refers to any major lipid presentin the blood. Serum lipids may be present in the blood either in freeform or as a part of a protein complex, e.g., a lipoprotein complex.Non-limiting examples of serum lipids may include triglycerides (TG),cholesterol, such as total cholesterol (TC), low density lipoproteincholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), verylow density lipoprotein cholesterol (VLDL-C) and intermediate-densitylipoprotein cholesterol (IDL-C).

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving a SCAP-associated disorder, is sufficient to effect treatment ofthe disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an iRNA that, when administered to a subjecthaving a SCAP-associated disorder, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the iRNA, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. iRNA employed in the methods of the presentinvention may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissue(or subcomponents thereof) or retinal tissue (or subcomponents thereof)derived from the subject.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of a SCAP gene.In one embodiment, the iRNA agent includes double stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of a SCAP gene in acell, such as a cell within a subject, e.g., a mammal, such as a humanhaving a SCAP-associated disorder, e.g., nonalcoholic fatty liverdisease (NAFLD) or nonalcoholic steatohepatitis (NASH). The dsRNAincludes an antisense strand having a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofa SCAP gene, The region of complementarity is about 30 nucleotides orless in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, or 18 nucleotides or less in length). Upon contact with a cellexpressing the SCAP gene, the iRNA inhibits the expression of the SCAPgene (e.g., a human, a primate, a non-primate, or a bird SCAP gene) byat least about 10% as assayed by, for example, a PCR or branched DNA(bDNA)-based method, or by a protein-based method, such as byimmunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a SCAPgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is between about 15 and about 23nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well known in the art that dsRNAslonger than about 21-23 nucleotides can serve as substrates for Dicer.As the ordinarily skilled person will also recognize, the region of anRNA targeted for cleavage will most often be part of a larger RNAmolecule, often an mRNA molecule. Where relevant, a “part” of an mRNAtarget is a contiguous sequence of an mRNA target of sufficient lengthto allow it to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target SCAP expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an antisense sequence. The sense strandsequence may be selected from the group of sequences provided in any oneof Tables 2, 3, 5 and 6, and the corresponding nucleotide sequence ofthe antisense strand of the sense strand may be selected from the groupof sequences of any one of Tables 2, 3, 5 and 6. In this aspect, one ofthe two sequences is complementary to the other of the two sequences,with one of the sequences being substantially complementary to asequence of an mRNA generated in the expression of a SCAP gene. As such,in this aspect, a dsRNA will include two oligonucleotides, where oneoligonucleotide is described as the sense strand (passenger strand) inany one of Tables 2, 3, 5 and 6, and the second oligonucleotide isdescribed as the corresponding antisense strand (guide strand) of thesense strand in any one of Tables 2, 3, 5 and 6. In one embodiment, thesubstantially complementary sequences of the dsRNA are contained onseparate oligonucleotides. In another embodiment, the substantiallycomplementary sequences of the dsRNA are contained on a singleoligonucleotide.

It will be understood that, although the sequences in Tables 2, 3, 5 and6 are described as modified and/or conjugated sequences, the RNA of theiRNA of the invention e.g., a dsRNA of the invention, may comprise anyone of the sequences set forth in any one of Tables 2, 3, 5 and 6 thatis un-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., (2001) EMBO J., 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided herein, dsRNAs described herein caninclude at least one strand of a length of minimally 21 nucleotides. Itcan be reasonably expected that shorter duplexes minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences provided herein, and differing in theirability to inhibit the expression of a SCAP gene by not more than about5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the fullsequence, are contemplated to be within the scope of the presentinvention.

In addition, the RNAs described herein identify a site(s) in a SCAPtranscript that is susceptible to RISC-mediated cleavage. As such, thepresent invention further features iRNAs that target within thissite(s). As used herein, an iRNA is said to target within a particularsite of an RNA transcript if the iRNA promotes cleavage of thetranscript anywhere within that particular site. Such an iRNA willgenerally include at least about 15 contiguous nucleotides from one ofthe sequences provided herein coupled to additional nucleotide sequencestaken from the region contiguous to the selected sequence in a SCAPgene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified herein represent effective targetsequences, it is contemplated that further optimization of inhibitionefficiency can be achieved by progressively “walking the window” onenucleotide upstream or downstream of the given sequences to identifysequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified herein,further optimization could be achieved by systematically either addingor removing nucleotides to generate longer or shorter sequences andtesting those sequences generated by walking a window of the longer orshorter size up or down the target RNA from that point. Again, couplingthis approach to generating new candidate targets with testing foreffectiveness of iRNAs based on those target sequences in an inhibitionassay as known in the art and/or as described herein can lead to furtherimprovements in the efficiency of inhibition. Further still, suchoptimized sequences can be adjusted by, e.g., the introduction ofmodified nucleotides as described herein or as known in the art,addition or changes in overhang, or other modifications as known in theart and/or discussed herein to further optimize the molecule (e.g.,increasing serum stability or circulating half-life, increasing thermalstability, enhancing transmembrane delivery, targeting to a particularlocation or cell type, increasing interaction with silencing pathwayenzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA agent as described herein can contain one or more mismatches tothe target sequence. In one embodiment, an iRNA as described hereincontains no more than 3 mismatches. If the antisense strand of the iRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch is not located in the center of the region ofcomplementarity. If the antisense strand of the iRNA contains mismatchesto the target sequence, it is preferable that the mismatch be restrictedto be within the last 5 nucleotides from either the 5′- or 3′-end of theregion of complementarity. For example, for a 23 nucleotide iRNA agentthe strand which is complementary to a region of a SCAP gene, generallydoes not contain any mismatch within the central 13 nucleotides. Themethods described herein or methods known in the art can be used todetermine whether an iRNA containing a mismatch to a target sequence iseffective in inhibiting the expression of a SCAP gene. Consideration ofthe efficacy of iRNAs with mismatches in inhibiting expression of a SCAPgene is important, especially if the particular region ofcomplementarity in a SCAP gene is known to have polymorphic sequencevariation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified. iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides. In still other embodiments of the invention,iRNAs of the invention can include not more than 5, 4, 3, 2 or 1modified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(.n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA of the invention can also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., (1991) Angewandte Chemie,International Edition, 30:613, and those disclosed by Sanghvi, Y S.,Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

An iRNA of the invention can also be modified to include one or morelocked nucleic acids (LNA). A locked nucleic acid is a nucleotide havinga modified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

An iRNA of the invention can also be modified to include one or morebicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modifiedby the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is anucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments an agent of theinvention may include one or more locked nucleic acids (LNA). A lockednucleic acid is a nucleotide having a modified ribose moiety in whichthe ribose moiety comprises an extra bridge connecting the 2′ and 4′carbons. In other words, an LNA is a nucleotide comprising a bicyclicsugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193). Examples of bicyclic nucleosides for use inthe polynucleotides of the invention include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, the antisense polynucleotide agents ofthe invention include one or more bicyclic nucleosides comprising a 4′to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides,include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

An iRNA of the invention can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of an iRNA of the invention include a 5′ phosphateor 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimicon the antisense strand of an RNAi agent. Suitable phosphate mimics aredisclosed in, for example US Patent Publication No. 2012/0157511, theentire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double-stranded RNAi agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO 2013/075035, filed on Nov. 16, 2012, the entirecontents of which are incorporated herein by reference. As shown hereinand in PCT Publication No. WO 2013/075035, a superior result may beobtained by introducing one or more motifs of three identicalmodifications on three consecutive nucleotides into a sense strandand/or antisense strand of an RNAi agent, particularly at or near thecleavage site. In some embodiments, the sense strand and antisensestrand of the RNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a GalNAc derivative ligand, for instance onthe sense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingactivity of the RNAi agent was superiorly enhanced.

Accordingly, the invention provides double stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., a SCAP gene) invivo. The RNAi agent comprises a sense strand and an antisense strand.Each strand of the RNAi agent may range from 12-30 nucleotides inlength. For example, each strand may be between 14-30 nucleotides inlength, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides inlength, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides inlength, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), and any combinations thereof. For example, TTcan be an overhang sequence for either end on either strand. Theoverhang can form a mismatch with the target mRNA or it can becomplementary to the gene sequences being targeted or can be anothersequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′ end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′ end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the RNAi agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the RNAi agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the RNAi agent further comprises aligand (preferably GalNAc₃).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3 ‘ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3’ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region which is at least 25 nucleotides in length, and thesecond strand is sufficiently complementary to a target mRNA along atleast 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradjacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking 0position may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or2′-fluoro. The strands can contain more than one modification. In oneembodiment, each residue of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the anti senese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′ end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):

5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′(I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′ n_(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ (Ib);5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n_(q) 3′ (Ic); or5′ n_(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n_(q) 3′ (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′ n_(p)-N_(a)-YYY-N_(a)-n_(q) 3′ (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′ n_(q)′-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n_(p)′3′ (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n_(p)′ 3′ (IIb);5′ n_(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n_(p)′ 3′ (IIc); or5′ n_(q)′-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n_(p)′3′ (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may berepresented by the formula:

5′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 3′ (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:

5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′n_(q)′ 5′ (IIIa)5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′n_(q)′ 5′ (IIIb)5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIc)5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n_(q)′ 5′(IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId)may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the invention is an agent selected from the group of agents listed inany one of Tables 2 and 3. These agents may further comprise a ligand.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., (1989) Proc.Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al.,(1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci.,660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let.,3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. AcidsRes., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanovet al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993)Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al.,(1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides,14:969-973), or adamantane acetic acid (Manoharan et al., (1995)Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al.,(1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) JPharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conujugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents In another aspect, the ligand is acell-permeation agent, preferably a helical cell-permeation agent.Preferably, the agent is amphipathic. An exemplary agent is a peptidesuch as tat or antennopedia. If the agent is a peptide, it can bemodified, including a peptidylmimetic, invertomers, non-peptide orpseudo-peptide linkages, and use of D-amino acids. The helical agent ispreferably an alpha-helical agent, which preferably has a lipophilic anda lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 27). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 28) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 29) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 30)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glyciosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In another embodiment,a carbohydrate conjugate for use in the compositions and methods of theinvention is selected from the group consisting of:

In one embodiment, the monosaccharide is an N-acetylgalactosamine, suchas

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Inanother embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more GalNAc (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A) P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherin one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CC or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a SCAP-associated disorder, e.g., Nonalcoholic FattyLiver Disease (NAFLD), e.g., nonalcoholic steatohepatitis (NASH)) can beachieved in a number of different ways. For example, delivery may beperformed by contacting a cell with an iRNA of the invention either invitro or in vivo. In vivo delivery may also be performed directly byadministering a composition comprising an iRNA, e.g., a dsRNA, to asubject. Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J. et al.,(2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J.et al. (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. etal., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007)Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al.(2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al. (2005) J Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A. et al.,(2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J Biol. Chem.279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. etal., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamerhas been shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O. et al., (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H. et al., (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al.(2003) J Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114),cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328;Pal, A. et al., (2005) Intl. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006)1 Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. etal., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNAforms a complex with cyclodextrin for systemic administration. Methodsfor administration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the SCAP gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,(1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a SCAP gene, e.g., a SCAP-associated disease, e.g.,Nonalcoholic Fatty Liver Disease (NAFLD), e.g., nonalcoholicsteatohepatitis (NASH).

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV),intramuscular (IM), or for subcutaneous (subQ) delivery. Another exampleis compositions that are formulated for direct delivery into the liver,e.g., by infusion into the liver, such as by continuous pump infusion.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a SCAP gene. In general, asuitable dose of an iRNA of the invention will be in the range of about0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. Typically, a suitable dose of an iRNA ofthe invention will be in the range of about 0.1 mg/kg to about 5.0mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

A repeat-dose regimine may include administration of a therapeuticamount of iRNA on a regular basis, such as every other day to once ayear. In certain embodiments, the iRNA is administered about once permonth to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as SCAP-associated disordersthat would benefit from reduction in the expression of SCAP. Such modelscan be used for in vivo testing of iRNA, as well as for determining atherapeutically effective dose. Suitable mouse models are known in theart and include, for example, an obese (ob/ob) mouse containing amutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes,52:1081-1089); a mouse containing homozygous knock-out of an LDLreceptor (LDLR−/− mouse; Ishibashi et al., (1993) J Clin Invest92(2):883-893); diet-induced artherosclerosis mouse model (Ishida etal., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoproteinlipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest.96(6):2555-2568).

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Feigner, P. L. et al., (1987) Proc. Natl.Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Banghamet al., (1965) M Mol. Biol. 23:238; Olson et al., (1979) Biochim.Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75:4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al.,(1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984)Endocrinol. 115:757. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer et al.,(1986) Biochim. Biophys. Acta 858:161. Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun.,147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleicacids rather than complex with them. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al. (1992) Journal of Controlled Release,19:269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel,(1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther.3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J.11:417.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside Gm′, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., (1987) FEBSLetters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., (1988), 85, 6949). U.S. Pat. No. 4,837,028 andWO 88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417,and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim.Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta1065:8). For certain cell lines, these liposomes containing conjugatedcationic lipids, are said to exhibit lower toxicity and provide moreefficient transfection than the DOTMA-containing compositions. Othercommercially available cationic lipid products include DMRIE andDMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (LifeTechnology, Inc., Gaithersburg, Md.). Other cationic lipids suitable forthe delivery of oligonucleotides are described in WO 98/39359 and WO96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting,vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research,18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al.(1987)Meth. Enzymol. 149:157-176; Straubinger, R. M. andPapahadjopoulos, D. (1983)Meth. Enzymol. 101:512-527; Wang, C. Y. andHuang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane(DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyptetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, di stearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in the tablebelow.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipidIonizable/Cationic Lipid conjugate Lipid: siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid: siRNA10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100) Lipid: siRNA10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl-4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid: siRNA 10:1 LNP121,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl) TechG1/DSPC/Cholesterol/PEG-DMG amino)ethyl)(2-hydroxydodecyl)amino)ethyl)50/10/38.5/1.5 piperazin-1-yl)ethylazanediyl)didodecan-2-ol Lipid: siRNA10:1 (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid: siRNA:11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/ GalNAc-PEG-DSG 50/10/35/4.5/0.5Lipid: siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid: siRNA:10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid: siRNA: 12:1LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid: siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid: siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid: siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-D SG 50/10/38.5/1.5 Lipid: siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described in PCT Publication No. WO2010/088537, the entire contents of which are hereby incorporated hereinby reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described in PCT Publication No. WO2010/054406, the entire contents of which are hereby incorporated hereinby reference.

C12-200 comprising formulations are described in PCT Publication No. WO2010/129709, the entire contents of which are hereby incorporated hereinby reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either aqueous phase, oily phase or itself as aseparate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories--surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating a SCAP-associated disorder. Examples of such agents include,but are not limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent. In addition, other substancescommonly used to protect the liver, such as silymarin, can also be usedin conjunction with the iRNAs described herein. Other agents useful fortreating liver diseases include telbivudine, entecavir, and proteaseinhibitors such as telaprevir and other disclosed, for example, in Tunget al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116,and 2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby SCAP expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VI. Methods for Inhibiting SCAP Expression

The present invention also provides methods of inhibiting expression ofa SCAP gene in a cell. The methods include contacting a cell with anRNAi agent, e.g., double stranded RNAi agent, in an amount effective toinhibit expression of SCAP in the cell, thereby inhibiting expression ofSCAP in the cell. In certain embodiments of the invention, SCAP isinhibited preferentially in liver cells.

Contacting of a cell with an iRNA, e.g., a double stranded RNAi agent,may be done in vitro or in vivo. Contacting a cell in vivo with the iRNAincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the iRNA. Combinations of in vitro and in vivomethods of contacting a cell are also possible. Contacting a cell may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In some embodiments, the targetingligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, or any otherligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a SCAP,” as used herein, includesinhibition of expression of any SCAP gene (such as, e.g., a mouse SCAPgene, a rat SCAP gene, a monkey SCAP gene, or a huma SCAP gene) as wellas variants or mutants of a SCAP gene that encode a SCAP protein. Thus,the SCAP gene may be a wild-type SCAP gene, a mutant SCAP gene, or atransgenic SCAP gene in the context of a genetically manipulated cell,group of cells, or organism.

“Inhibiting expression of a SCAP gene” includes any level of inhibitionof a SCAP gene, e.g., at least partial suppression of the expression ofa SCAP gene, such as an inhibition by at least about 20%. In certainembodiments, inhibition is by at least about 25%, at least about 30%, atleast about 35%,at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of a SCAP gene may be assessed based on the level of anyvariable associated with SCAP gene expression, e.g., SCAP mRNA level orSCAP protein level. The expression of a SCAP may also be assessedindirectly based on the levels of a serum lipid, a triglyceride,cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and totalcholesterol), or free fatty acids. The expression of a SCAP may also beassessed indirectly based on the level of SREPB levels and/or the PNPLA3levels.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more of these variables compared with a control level.The control level may be any type of control level that is utilized inthe art, e.g., a pre-dose baseline level, or a level determined from asimilar subject, cell, or sample that is untreated or treated with acontrol (such as, e.g., buffer only control or inactive agent control).

In certain embodiments, surrogate markers can be used to detectinhibition of SCAP. For example, effective treatment of aSCAP-associated disorder, e.g., a liver disorder, as demonstrated byacceptable diagnostic and monitoring criteria with an agent to reduceSCAP expression can be understood to demonstrate a clinically relevantreduction in SCAP.

In some embodiments of the methods of the invention, expression of aSCAP gene is inhibited by at least 20%, a 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level ofdetection of the assay. In certain embodiments, the methods include aclinically relevant inhibition of expression of SCAP, e.g. asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of SCAP.

Inhibition of the expression of a SCAP gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a SCAP gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an iRNA of theinvention, or by administering an iRNA of the invention to a subject inwhich the cells are or were present) such that the expression of a SCAPgene is inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas not or have not been so treated (control cell(s) not treated with aniRNA or not treated with an iRNA targeted to the gene of interest). Thedegree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a SCAP gene may beassessed in terms of a reduction of a parameter that is functionallylinked to SCAP gene expression, e.g., SCAP protein expression or SCAPsignaling pathways. SCAP gene silencing may be determined in any cellexpressing SCAP, either endogenous or heterologous from an expressionconstruct, and by any assay known in the art.

Inhibition of the expression of a SCAP protein may be manifested by areduction in the level of the SCAP protein that is expressed by a cellor group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above, for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a SCAP gene includes a cell or group ofcells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of SCAP mRNA that is expressed by a cell or group of cells maybe determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of SCAP in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the SCAP gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays, northern blotting, in situ hybridization, and microarrayanalysis. Circulating SCAP mRNA may be detected using methods thedescribed in PCT Publication WO2012/177906, the entire contents of whichare hereby incorporated herein by reference. In some embodiments, thelevel of expression of SCAP is determined using a nucleic acid probe.The term “probe”, as used herein, refers to any molecule that is capableof selectively binding to a specific SCAP. Probes can be synthesized byone of skill in the art, or derived from appropriate biologicalpreparations. Probes may be specifically designed to be labeled.Examples of molecules that can be utilized as probes include, but arenot limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to SCAPmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of SCAP mRNA.

An alternative method for determining the level of expression of SCAP ina sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of SCAP isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System)or the Dual-Glo® Luciferase assay in Example 2.

The expression levels of SCAP mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of SCAP expressionlevel may also comprise using nucleic acid probes in solution.

In some embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCRmethod is described and exemplified in the Examples presented herein.Such methods can also be used for the detection of SCAP nucleic acids,SREBP nucleic acids or PNPLA3 nucleic acids.

The level of SCAP protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like. Such assays can also beused for the detection of proteins indicative of the presence orreplication of SCAP proteins, SREBP protein or PNPLA3 protein.

In some embodiments, the efficacy of the methods of the invention in thetreatment of a SCAP-related disease is assessed by a decrease in SCAPmRNA level (by liver biopsy).

In some embodiments of the methods of the invention, the iRNA isadministered to a subject such that the iRNA is delivered to a specificsite within the subject. The inhibition of expression of SCAP may beassessed using measurements of the level or change in the level of SCAPmRNA or SCAP protein in a sample derived from a specific site within thesubject, e.g., the liver. In certain embodiments, the methods include aclinically relevant inhibition of expression of SCAP, e.g. asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of SCAP.

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

VII. Methods of Treating or Preventing SCAP-Associated Diseases

The present invention also provides methods of using an iRNA of theinvention and/or a composition containing an iRNA of the invention toreduce and/or inhibit SCAP expression in a cell. The methods includecontacting the cell with a dsRNA of the invention and maintaining thecell for a time sufficient to obtain degradation of the mRNA transcriptof a SCAPgene, thereby inhibiting expression of the SCAP gene in thecell. Reduction in gene expression can be assessed by any methods knownin the art. For example, a reduction in the expression of SCAP may bedetermined by determining the mRNA expression level of SCAP usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR; by determining the protein level of SCAP usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques. A reduction in the expression ofSCAP may also be assessed indirectly by measuring a decrease inbiological activity of SCAP, e.g., a decrease in the level of serumlipid, triglycerides, cholesterol and/or free fatty acids, a decrease inthe level of SREPB or PNPLA3.

In the methods of the invention the cell may be contacted in vitro or invivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses a SCAPgene. A cell suitable for use in themethods of the invention may be a mammalian cell, e.g., a primate cell(such as a human cell or a non-human primate cell, e.g., a monkey cellor a chimpanzee cell), a non-primate cell (such as a cow cell, a pigcell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbitcell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dogcell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell,or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), ora whale cell. In one embodiment, the cell is a human cell, e.g., a humanliver cell.

SCAP expression is inhibited in the cell by at least about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or about 100%. In preferred embodiments, SCAP expression is inhibited byat least 20%.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the SCAP gene of the mammal to be treated. When theorganism to be treated is a mammal such as a human, the composition canbe administered by any means known in the art including, but not limitedto oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection. In certain embodiments, the compositions areadministered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof SCAP, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of a SCAP gene in a mammal. The methodsinclude administering to the mammal a composition comprising a dsRNAthat targets a SCAP gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the SCAP gene, thereby inhibiting expression of the SCAPgene in the cell. Reduction in gene expression can be assessed by anymethods known it the art and by methods, e.g. qRT-PCR, described herein.Reduction in protein production can be assessed by any methods known itthe art and by methods, e.g. ELISA, described herein. In one embodiment,a puncture liver biopsy sample serves as the tissue material formonitoring the reduction in SCAP gene and/or protein expression.

The present invention further provides methods of treatment of a subjectin need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction and/or inhibition of SCAPexpression, in a therapeutically effective amount of an iRNA targeting aSCAP gene or a pharmaceutical composition comprising an iRNA targeting aSCAP gene.

The present invention also provides methods of decreasing plasmatriglyceride levels in a subject. The methods include administering aniRNA of the invention to a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of SCAP expression, in atherapeutically effective amount of an iRNA targeting a SCAP gene or apharmaceutical composition comprising an iRNA targeting a SCAP gene.

In addition, the present invention provides methods of inhibiting theprogression of NAFLD in a subject, such as the progression of steatosisto NASH in a subject having steatosis or a subject at risk of developingsteatosis, e.g., a subject having inusin resistance or an obese subject;or the progression of NASH to cirrhosis in a subject having NASH or asubject at risk of developing cirrhosis. The methods includeadministering to the subject a therapeutically effective amount of anyof the dsRNAs or the pharmaceutical composition provided herein, therebyinhibiting the progeression of NAFLD in the subject.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of SCAPgene expression are those having a SCAP-associated disorder. The term“SCAP-associated disease” includes a disease, disorder or condition thatwould benefit from a decrease in SCAP gene expression, replication, orprotein activity. Non-limiting examples of SCAP-associated diseasesinclude, for example, fatty liver (steatosis), nonalcoholicsteatohepatitis (NASH), cirrhosis of the liver, accumulation of fat inthe liver, inflammation of the liver, hepatocellular necrosis, liverfibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD),hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulindependent diatebes mellitus, coronary heart disease, and cerebrovasculardisease. In another embodiment, the SCAP-associated disease isnonalcoholic fatty liver disease (NAFLD). In another embodiment, theSCAP-associated disease is nonalcoholic steatohepatitis (NASH). Inanother embodiment, the SCAP-associated disease is liver cirrhosis. Inanother embodiment, the SCAP-associated disease is insulin resistance.In another embodiment, the SCAP-associated disease is not insulinresistance. In one embodiment, the SCAP-associated disease is obesity.

The invention further provides methods for the use of an iRNA or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of SCAP expression, e.g.,a subject having a SCAP-associated disorder, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, in certain embodiments, an iRNA targeting SCAP is administeredin combination with, e.g., an agent useful in treating a SCAP-associateddisorder as described elsewhere herein. For example, additional agentssuitable for treating a subject that would benefit from reduction inSCAP expression, e.g., a subject having a SCAP-associated disorder, mayinclude agents that lower one or more serum lipids. Non-limitingexamples of such agents may include cholesterol synthesis inhibitors,such as HMGCR inhibitors, e.g., statins. Statins may includeatorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor),lovastatin extended-release (Altoprev), pitavastatin (Livalo),pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin(Zocor). Other agents useful in treating a SCAP-associated disorder mayinclude bile sequestering agents, such as cholestyramine and otherresins; VLDL secretion inhibitors, such as niacin; lipophilicantioxidants, such as Probucol; acyl-CoA cholesterol acyl transferaseinhibitors; farnesoid X receptor antagonists; sterol regulatory bindingprotein cleavage activating protein (SCAP) activators; microsomaltriglyceride transfer protein (MTP) inhibitors; ApoE-related peptide;and therapeutic antibodies against SCAP. The additional therapeuticagents may also include agents that raise high density lipoprotein(HDL), such as cholesteryl ester transfer protein (CETP) inhibitors.Furthermore, the additional therapeutic agents may also include dietarysupplements, e.g., fish oil. The iRNA and additional therapeutic agentsmay be administered at the same time and/or in the same combination,e.g., parenterally, or the additional therapeutic agent can beadministered as part of a separate composition or at separate timesand/or by another method known in the art or described herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target SCAP gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24hours, 28, 32, or about 36 hours. In one embodiment, expression of thetarget SCAP gene is decreased for an extended duration, e.g., at leastabout two, three, four days or more, e.g., about one week, two weeks,three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featuredherein specifically target RNAs (primary or processed) of the targetSCAP gene. Compositions and methods for inhibiting the expression ofthese genes using iRNAs can be prepared and performed as describedherein.

Administration of the dsRNA according to the methods of the inventionmay result in a reduction of the severity, signs, symptoms, and/ormarkers of such diseases or disorders in a patient with aSCAP-associated disorder. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of a SCAP-associated disorder may beassessed, for example, by periodic monitoring of one or more serum lipidlevels. Comparisons of the later readings with the initial readingsprovide a physician an indication of whether the treatment is effective.It is well within the ability of one skilled in the art to monitorefficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. In connection with theadministration of an iRNA targeting SCAP or pharmaceutical compositionthereof, “effective against” a SCAP-associated disorder indicates thatadministration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as a improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating SCAP-associated disorders and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example the Child-Pugh score (sometimes the Child-Turcotte-Pughscore). Any positive change resulting in e.g., lessening of severity ofdisease measured using the appropriate scale, represents adequatetreatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg to about 200 mg/kg.

The iRNA can be administered by intravenous infusion over a period oftime, on a regular basis. In certain embodiments, after an initialtreatment regimen, the treatments can be administered on a less frequentbasis. Administration of the iRNA can reduce SCAP levels, e.g., in acell, tissue, blood, urine or other compartment of the patient by atleast about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or at least about 99% or more. In a preferredembodiment, administration of the iRNA can reduce SCAP levels, e.g., ina cell, tissue, blood, urine or other compartment of the patient by atleast 20%.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired daily dose of iRNA to a subject. The injections may berepeated over a period of time. The administration may be repeated on aregular basis. In certain embodiments, after an initial treatmentregimen, the treatments can be administered on a less frequent basis. Arepeat-dose regimine may include administration of a therapeutic amountof iRNA on a regular basis, such as every other day or to once a year.In certain embodiments, the iRNA is administered about once per month toabout once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1. iRNA Design, Synthesis, Selection, and In VitroEvaluation

This Example describes methods for the design, synthesis, selection, andin vitro evaluation of SCAP iRNA agents.

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Bioinformatics

A set of siRNA agents targeting the huma SCAP gene, “Homo sapiens SREBFchaperone” (human: NCBI refseqID NM_012235; NCBI GeneID: 22937), as wellas toxicology-species SCAP orthologs (cynomolgus monkey: XM_005546963;mouse: NM_001103162; rat: NM_001100966) were designed using custom R andPython scripts. The human NM_012235 REFSEQ mRNA, version 2, has a lengthof 4255 bases. The rationale and method for the set of siRNA designs isas follows: the predicted efficacy for every potential 19mer siRNA fromposition 10 through position 4255 was determined with a linear modelderived the direct measure of mRNA knockdown from more than 20,000distinct siRNA designs targeting a large number of vertebrate genes.Subsets of the SCAP siRNAs were designed with perfect or near-perfectmatches between human and cynomolgus monkey. A further subset wasdesigned with perfect or near-perfect matches to mouse and rat SCAPorthologs. A further subset was designed with perfect or near-perfectmatches to human, cynomolgus monkey and mouse SCAP orthologs. A furthersubset was designed with perfect or near-perfect matches to human,cynomolgus monkey, mouse, and rat SCAP orthologs. For each strand of thesiRNA, a custom Python script was used in a brute force search tomeasure the number and positions of mismatches between the siRNA and allpotential alignments in the target species transcriptome. Extra weightwas given to mismatches in the seed region, defined here as positions2-9 of the antisense oligonucleotide, as well the cleavage site of thesiRNA, defined here as positions 10-11 of the antisense oligonucleotide.The relative weight of the mismatches was 2.8; 1.2:1 for seedmismatches, cleavage site, and other positions up through antisenseposition 19. Mismatches in the first position were ignored. Aspecificity score was calculated for each strand by summing the value ofeach weighted mismatch. Preference was given to siRNAs whose antisensescore in human was >=2.2 and predicted efficacy was >=50% knockdown ofthe SCAP transcript.

Synthesis of SCAP Sequences Synthesis of SCAP Single Strands andDuplexes

SCAP siRNA sequences were synthesized at 1 μmol scale on Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500° A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F, 2′-O-Methyl, RNA, DNA and other modifiednucleosides were introduced in the sequences using the correspondingphosphoramidites. Synthesis of 3′ GalNAc conjugated single strands wasperformed on a GalNAc modified CPG support. Custom CPG universal solidsupport was used for the synthesis of antisense single strands. Couplingtime for all phosphoramidites (100 mM in acetonitrile) was 5 minutesemploying 5-Ethylthio-1H-tetrazole (ETT) as activator (0.6 M inacetonitrile). Phosphorothioate linkages were generated using a 50 mMsolution of 3-((Dimethylamino-methylidene) amino)-3H-1, 2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass.,USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3minutes. All sequences were synthesized with final removal of the DMTgroup (“DMT off”).

Upon completion of the solid phase synthesis, single strands werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagent at 60° C. for 20minutes. For sequences containing 2′ ribo residues (2′-OH) that areprotected with tert-butyl dimethyl silyl (TBDMS) group, a second stepdeprotection was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To the methylamine deprotection solution, 200 uL ofdimethyl sulfoxide (DMSO) and 300 ul TEA.3HF reagent was added and thesolution was incubated for additional 20 minutes at 60° C. At the end ofcleavage and deprotection step, the synthesis plate was allowed to cometo room temperature and was precipitated by addition of 1 mL ofacetontile:ethanol mixture (9:1). The plates were cooled at −80° C. for2 hours and the supernatant decanted carefully with the aid of amulti-channel pipette. The oligonucleotide pellet was re-suspended in 20mM NaOAc buffer and were desalted using a 5 mL HiTrap size exclusioncolumn (GE Healthcare) on an AKTA Purifier System equipped with an A905autosampler and a Frac 950 fraction collector. Desalted samples werecollected in 96 well plates. Samples from each sequence were analyzed byLC-MS to confirm the identity, UV (260 nm) for quantification and aselected set of samples by IEX chromatography to determine purity.

Annealing of SCAP single strands was performed on a Tecan liquidhandling robot. Equimolar mixture of sense and antisense single strandswere combined and annealed in 96 well plates. After combining thecomplementary single strands, the 96 well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10 uM in 1×PBS and then submitted for in vitroscreening assays.

A detailed list of the modified SCAP sense and antisense strandsequences is shown in Table 2 and a detailed list of the unmodified SCAPsense and antisense strand sequences is shown in Table 3.

In Vitro Screening: Cell Culture and Transfections:

Primary Mouse Hepatocytes (PMH) (GIBCO) and Primary Cyno Hepatocytes(PCH) (Celsis) were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μlof Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. Forty μl of William's EMedium (Life Tech) containing about 5×10³ cells were then added to thesiRNA mixture. Cells were incubated for 24 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADS (Invitrogen, cat #61012). Fifty μ1 of Lysis/BindingBuffer and 25 μl of Lysis Buffer containing 3 μL of magnetic beads wereadded to the plate with cells. Plates were incubated on anelectromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand supernatant was removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1μl Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction were added to RNA isolated per well.Plates were sealed, mixed and incubated on an electromagnetic shaker for10 minutes at room temperature, and then incubated at 37° C. for 2hours. Plates were then incubated at 81° C. for 8 minutes.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μl of humanGAPDH TaqMan Probe (4326317E), 0.5 μl huma SCAP (Hs00168352_ml), and 5μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well ina 384 well plates (Roche cat #04887301001). Real time PCR was done in aLightCycler480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay.Each duplex was tested in at least two times and data were normalized tocells transfected with a non-targeting control siRNA.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with a non-targeting control siRNA. The results from theassays are shown in Table 4.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]- 4-hydroxyprolinolHyp-(GalNAc-alkyl)3 dT 2′-deoxythymidine-3′-phosphate dC2′-deoxycytidine-3′-phosphate P Phosphate VP Vinyl-phosphate

TABLE 2 SCAP Modified Sequences SEQ SEQ SEQ Duplex ID ID ID NameSense Sequence (5′ to 3′) NO Antisense Sequence (5′ to 3′) NOmRNA target sequence NO AD-68570 usgsgcaaCfaAfGfGfugacuuagcaL96  31usGfscuaAfgUfCfaccuUfgUfugccasusc  32 GAUGGCAACAAGGUGACUUAGCC  33AD-68572 cscscugaCfuGfAfAfaggcuucguaL96  34usAfscgaAfgCfCfuuucAfgUfcagggsusc  35 GACCCUGACUGAAAGGCUUCGUG  36AD-68565 gsgscuucGfuGfAfGfaagauaucuaL96  37usAfsgauAfuCfUfucucAfcGfaagccsusu  38 AAGGCUUCGUGAGAAGAUAUCUC  39AD-68552 usgsgcagCfaAfGfGfugacuucggaL96  40usCfscgaAfgUfCfaccuUfgCfugccasusc  41 GAUGGCAGCAAGGUGACUUCGGC  42AD-68533 asusgaccCfuGfAfCfugaaaggcuaL96  43usAfsgccUfuUfCfagucAfgGfgucauscsc  44 GGAUGACCCUGACUGAAAGGCUG  45AD-68543 asgsgaacAfgGfAfCfcuguggaauuL96  46asAfsuucCfaCfAfggucCfuGfuuccusgsg  47 CCAGGAACAGGACCUGUGGAAUU  48AD-68541 gsusccagCfaGfAfUfauuugugaaaL96  49usUfsucaCfaAfAfuaucUfgCfuggacsasu  50 AUGUCCAGCAGAUAUUUGUGAAG  51AD-68555 csasguagAfuGfUfAfuuucguucapL96  52usUfsgaaCfgAfAfauacAfuCfuacugscsc  53 GGCAGUAGAUGUAUUUCGUUCAC  54AD-68540 csascgugCfuGfAfGfagacagcucuL96  55asGfsagcUfgUfCfucucAfgCfacgugsgsu  56 ACCACGUGCUGAGAGACAGCUCU  57AD-68551 ususccauGfcUfGfAfuccugacauaL96  58usAfsuguCfaGfGfaucaGfcAfuggaasgsc  59 GCUUCCAUGCUGAUCCUGACAUC  60AD-68549 csasccagGfaAfGfAfggauggucuaL96  61usAfsgacCfaUfCfcucuUfcCfuggugsusa  62 UACACCAGGAAGAGGAUGGUCUC  63AD-68542 csasggaaGfaGfGfAfuggucuccuaL96  64usAfsggaGfaCfCfauccUfcUfuccugsgsu  65 ACCAGGAAGAGGAUGGUCUCCUA  66AD-68529 asuscaucUfuGfUfUfugccuacauaL96  67usAfsuguAfgGfCfaaacAfaGfaugausgsu  68 ACAUCAUCUUGUUUGCCUACAUC  69AD-68536 csasucuuGfuUfUfGfccuacaucuaL96  70usAfsgauGfuAfGfgcaaAfcAfagaugsasu  71 AUCAUCUUGUUUGCCUACAUCUA  72AD-68539 uscsuuguUfuGfCfCfuacaucuacuL96  73asGfsuagAfuGfUfaggcAfaAfcaagasusg  74 CAUCUUGUUUGCCUACAUCUACU  75AD-68530 gsasagauCfgAfCfAfuggucaaguaL96  76usAfscuuGfaCfCfauguCfgAfucuucscsg  77 CGGAAGAUCGACAUGGUCAAGUC  78AD-68569 gscsucacCfaAfGfUfcagugguauaL96  79usAfsuacCfaCfUfgacuUfgGfugagcsasc  80 GUGCUCACCAAGUCAGUGGUAUC  81AD-68564 csasccaaGfuCfAfGfugguaucaaaL96  82usUfsugaUfaCfCfacugAfcUfuggugsasg  83 CUCACCAAGUCAGUGGUAUCAAC  84AD-68547 csuscaauGfgCfGfGfcgagauuuuaL96  85usAfsaaaUfcUfCfgccgCfcAfuugagsgsg  86 CCCUCAAUGGCGGCGAGAUUUUC  87AD-68548 usasccuuGfuGfGfUfgguuauuggaL96  88usCfscaaUfaAfCfcaccAfcAfagguasgsg  89 CCUACCUUGUGGUGGUUAUUGGG  90AD-68544 usgsguuaUfuGfGfGfuuagagaauaL96  91usAfsuucUfcUfAfacccAfaUfaaccascsc  92 GGUGGUUAUUGGGUUAGAGAAUG  93AD-68557 gsusuauuGfgGfUfUfagagaauguaL96  94usAfscauUfcUfCfuaacCfcAfauaacscsa  95 UGGUUAUUGGGUUAGAGAAUGUG  96AD-68535 gsusuagaGfaAfUfGfuguuggugcuL96  97asGfscacCfaAfCfacauUfcUfcuaacscsc  98 GGGUUAGAGAAUGUGUUGGUGCU  99AD-68567 csusgacuGfaAfAfGfgcuucgugaaL96 100usUfscacGfaAfGfccuuUfcAfgucagsgsg 101 CCCUGACUGAAAGGCUUCGUGAG 102AD-68538 ascscguuGfuCfUfGfgauuggcauaL96 103usAfsugcCfaAfUfccagAfcAfacggusgsc 104 GCACCGUUGUCUGGAUUGGCAUC 105AD-68532 ascsuuugGfaGfGfAfaauuguccuuL96 106asAfsggaCfaAfUfuuccUfcCfaaagususc 107 GAACUUUGGAGGAAAUUGUCCUU 108AD-68528 ususuggaGfgAfAfAfuuguccuucaL96 109usGfsaagGfaCfAfauuuCfcUfccaaasgsu 110 ACUUUGGAGGAAAUUGUCCUUCC 111AD-68553 gsgsagagCfuGfGfGfaacgacuuuaL96 112usAfsaagUfcGfUfucccAfgCfucuccsusg 113 CAGGAGAGCUGGGAACGACUUUC 114AD-68554 asgscuggGfaAfCfGfacuuucagauL96 115asUfscugAfaAfGfucguUfcCfcagcuscsu 116 AGAGCUGGGAACGACUUUCAGAU 117AD-68545 csgsacuuUfcAfGfAfuggugggaaaL96 118usUfsuccCfaCfCfaucuGfaAfagucgsusu 119 AACGACUUUCAGAUGGUGGGAAG 120AD-68550 ascscugcUfuAfAfUfugacaccaaaL96 121usUfsuggUfgUfCfaauuAfaGfcaggusgsa 122 UCACCUGCUUAAUUGACACCAAC 123AD-68546 gscsuuaaUfuGfAfCfaccaacuuuuL96 124asAfsaagUfuGfGfugucAfaUfuaagcsasg 125 CUGCUUAAUUGACACCAACUUUU 126AD-68571 gsgsgagcCfaAfGfAfccauacucuaL96 127usAfsgagUfaUfGfgucuUfgGfcucccsusg 128 CAGGGAGCCAAGACCAUACUCUA 129AD-68556 ascsccauCfaCfAfGfcccugaaagaL96 130usCfsuuuCfaGfGfgcugUfgAfugggususu 131 AAACCCAUCACAGCCCUGAAAGC 132AD-68568 ascsagcuGfuGfUfAfcauugaucaaL96 133usUfsgauCfaAfUfguacAfcAfgcugusgsa 134 UCACAGCUGUGUACAUUGAUCAG 135AD-68563 asgscuguGfuAfCfAfuugaucagaaL96 136usUfscugAfuCfAfauguAfcAfcagcusgsu 137 ACAGCUGUGUACAUUGAUCAGAC 138AD-68560 csusgccuGfuGfGfGfauguacuaaaL96 139usUfsuagUfaCfAfucccAfcAfggcagsasu 140 AUCUGCCUGUGGGAUGUACUAAC 141AD-68562 asgsuggcCfuGfGfAfugacuucauaL96 142usAfsugaAfgUfCfauccAfgGfccacusasc 143 GUAGUGGCCUGGAUGACUUCAUC 144AD-68559 csusccuuUfuGfGfGfaccuaaacuaL96 145usAfsguuUfaGfGfucccAfaAfaggagsasc 146 GUCUCCUUUUGGGACCUAAACUA 147AD-68561 cscsuuuuGfgGfAfCfcuaaacuauaL96 148usAfsuagUfuUfAfggucCfcAfaaaggsasg 149 CUCCUUUUGGGACCUAAACUAUG 150AD-68558 asasgcuuGfgGfUfGfucaucucagaL96 151usCfsugaGfaUfGfacacCfcAfagcuusgsc 152 GCAAGCUUGGGUGUCAUCUCAGA 153AD-68531 asgsggcuGfuGfUfCfuccuuuuggaL96 154usCfscaaAfaGfGfagacAfcAfgcccusgsg 155 CCAGGGCUGUGUCUCCUUUUGGG 156AD-68537 gscsugccAfuUfGfUfcugcaacuuuL96 157asAfsaguUfgCfAfgacaAfuGfgcagcsgsu 158 ACGCUGCCAUUGUCUGCAACUUU 159AD-68527 csusgccaUfuGfUfCfugcaacuuuaL96 160usAfsaagUfuGfCfagacAfaUfggcagscsg 161 CGCUGCCAUUGUCUGCAACUUUG 162AD-68534 asascuuuGfgCfAfGfugagcucagaL96 163usCfsugaGfcUfCfacugCfcAfaaguusgsc 164 GCAACUUUGGCAGUGAGCUCAGC 165AD-68566 uscsuccuGfaCfUfGfuaauaauaaaL96 166usUfsuauUfaUfUfacagUfcAfggagascsa 167 UGUCUCCUGACUGUAAUAAUAAA 168

TABLE 3 SCAP Unmodified Sequences SEQ SEQ Duplex Sense Sequence IDPosition in Antisense Sequence ID Name (5′ to 3′) NO NM_012235.2(5′ to 3′) NO AD-68570 UGGCAACAAGGUGACUUAGCA 169 135-155UGCUAAGUCACCUUGUUGCCAUC 170 AD-68572 CCCUGACUGAAAGGCUUCGUA 171 164-184UACGAAGCCUUUCAGUCAGGGUC 172 AD-68565 GGCUUCGUGAGAAGAUAUCUA 173 176-196UAGAUAUCUUCUCACGAAGCCUU 174 AD-68552 UGGCAGCAAGGUGACUUCGGA 175 230-250UCCGAAGUCACCUUGCUGCCAUC 176 AD-68533 AUGACCCUGACUGAAAGGCUA 177 256-276UAGCCUUUCAGUCAGGGUCAUCC 178 AD-68543 AGGAACAGGACCUGUGGAAUU 179 405-425AAUUCCACAGGUCCUGUUCCUGG 180 AD-68541 GUCCAGCAGAUAUUUGUGAAA 181 532-552UUUCACAAAUAUCUGCUGGACAU 182 AD-68555 CAGUAGAUGUAUUUCGUUCAA 183 587-607UUGAACGAAAUACAUCUACUGCC 184 AD-68540 CACGUGCUGAGAGACAGCUCU 185 649-669AGAGCUGUCUCUCAGCACGUGGU 186 AD-68551 UUCCAUGCUGAUCCUGACAUA 187 808-828UAUGUCAGGAUCAGCAUGGAAGC 188 AD-68549 CACCAGGAAGAGGAUGGUCUA 189 933-953UAGACCAUCCUCUUCCUGGUGUA 190 AD-68542 CAGGAAGAGGAUGGUCUCCUA 191 936-956UAGGAGACCAUCCUCUUCCUGGU 192 AD-68529 AUCAUCUUGUUUGCCUACAUA 193 1132-1152UAUGUAGGCAAACAAGAUGAUGU 194 AD-68536 CAUCUUGUUUGCCUACAUCUA 195 1134-1154UAGAUGUAGGCAAACAAGAUGAU 196 AD-68539 UCUUGUUUGCCUACAUCUACU 197 1136-1156AGUAGAUGUAGGCAAACAAGAUG 198 AD-68530 GAAGAUCGACAUGGUCAAGUA 199 1167-1187UACUUGACCAUGUCGAUCUUCCG 200 AD-68569 GCUCACCAAGUCAGUGGUAUA 201 1248-1268UAUACCACUGACUUGGUGAGCAC 202 AD-68564 CACCAAGUCAGUGGUAUCAAA 203 1251-1271UUUGAUACCACUGACUUGGUGAG 204 AD-68547 CUCAAUGGCGGCGAGAUUUUA 205 1282-1302UAAAAUCUCGCCGCCAUUGAGGG 206 AD-68548 UACCUUGUGGUGGUUAUUGGA 207 1306-1326UCCAAUAACCACCACAAGGUAGG 208 AD-68544 UGGUUAUUGGGUUAGAGAAUA 209 1316-1336UAUUCUCUAACCCAAUAACCACC 210 AD-68557 GUUAUUGGGUUAGAGAAUGUA 211 1318-1338UACAUUCUCUAACCCAAUAACCA 212 AD-68535 GUUAGAGAAUGUGUUGGUGCU 213 1326-1346AGCACCAACACAUUCUCUAACCC 214 AD-68567 CUGACUGAAAGGCUUCGUGAA 215 166-186UUCACGAAGCCUUUCAGUCAGGG 216 AD-68538 ACCGUUGUCUGGAUUGGCAUA 217 1825-1845UAUGCCAAUCCAGACAACGGUGC 218 AD-68532 ACUUUGGAGGAAAUUGUCCUU 219 2124-2144AAGGACAAUUUCCUCCAAAGUUC 220 AD-68528 UUUGGAGGAAAUUGUCCUUCA 221 2126-2146UGAAGGACAAUUUCCUCCAAAGU 222 AD-68553 GGAGAGCUGGGAACGACUUUA 223 2748-2768UAAAGUCGUUCCCAGCUCUCCUG 224 AD-68554 AGCUGGGAACGACUUUCAGAU 225 2752-2772AUCUGAAAGUCGUUCCCAGCUCU 226 AD-68545 CGACUUUCAGAUGGUGGGAAA 227 2761-2781UUUCCCACCAUCUGAAAGUCGUU 228 AD-68550 ACCUGCUUAAUUGACACCAAA 229 2875-2895UUUGGUGUCAAUUAAGCAGGUGA 230 AD-68546 GCUUAAUUGACACCAACUUUU 231 2879-2899AAAAGUUGGUGUCAAUUAAGCAG 232 AD-68571 GGGAGCCAAGACCAUACUCUA 233 3433-3453UAGAGUAUGGUCUUGGCUCCCUG 234 AD-68556 ACCCAUCACAGCCCUGAAAGA 235 3495-3515UCUUUCAGGGCUGUGAUGGGUUU 236 AD-68568 ACAGCUGUGUACAUUGAUCAA 237 3517-3537UUGAUCAAUGUACACAGCUGUGA 238 AD-68563 AGCUGUGUACAUUGAUCAGAA 239 3519-3539UUCUGAUCAAUGUACACAGCUGU 240 AD-68560 CUGCCUGUGGGAUGUACUAAA 241 3576-3596UUUAGUACAUCCCACAGGCAGAU 242 AD-68562 AGUGGCCUGGAUGACUUCAUA 243 3676-3696UAUGAAGUCAUCCAGGCCACUAC 244 AD-68559 CUCCUUUUGGGACCUAAACUA 245 3816-3836UAGUUUAGGUCCCAAAAGGAGAC 246 AD-68561 CCUUUUGGGACCUAAACUAUA 247 3818-3838UAUAGUUUAGGUCCCAAAAGGAG 248 AD-68558 AAGCUUGGGUGUCAUCUCAGA 249 3867-3887UCUGAGAUGACACCCAAGCUUGC 250 AD-68531 AGGGCUGUGUCUCCUUUUGGA 251 3911-3931UCCAAAAGGAGACACAGCCCUGG 252 AD-68537 GCUGCCAUUGUCUGCAACUUU 253 4021-4041AAAGUUGCAGACAAUGGCAGCGU 254 AD-68527 CUGCCAUUGUCUGCAACUUUA 255 4022-4042UAAAGUUGCAGACAAUGGCAGCG 256 AD-68534 AACUUUGGCAGUGAGCUCAGA 257 4036-4056UCUGAGCUCACUGCCAAAGUUGC 258 AD-68566 UCUCCUGACUGUAAUAAUAAA 259 4097-4117UUUAUUAUUACAGUCAGGAGACA 260

TABLE 4 SCAP Single Dose Screen in Primary Mouse and Primary CynoHepatocytes Data are expressed as percent message remaining relative toAD-1955 non-targeting control. Primary Mouse Hepatocytes Primary CynoHepatocytes 10 nM 10 nM 0.1 nM 0.1 nM 10 nM 10 nM 0.1 nM 0.1 nM DuplexIDAvg SD Avg SD Avg SD Avg SD AD-68570.1 41.7 4.9 99.7 4.7 80.9 9.5 100.68.1 AD-68572.1 35.3 4.7 89.7 6.0 45.7 5.3 89.0 6.4 AD-68565.1 28.2 5.288.8 11.2 34.5 3.5 62.3 5.1 AD-68552.1 111.9 29.8 119.5 13.4 39.0 4.591.6 4.4 AD-68533.1 30.1 7.2 60.7 6.1 34.6 5.7 80.5 7.5 AD-68543.1 72.917.4 94.1 13.6 32.9 5.4 60.8 6.5 AD-68541.1 42.0 5.5 71.0 9.9 20.6 2.744.3 2.9 AD-68555.1 102.9 16.9 106.4 5.5 20.2 3.4 46.2 6.1 AD-68540.150.6 7.6 73.0 4.2 42.0 5.1 83.7 3.2 AD-68551.1 93.4 13.0 96.2 6.1 26.12.2 44.5 7.2 AD-68549.1 74.1 4.2 83.1 6.6 42.2 7.1 88.8 7.4 AD-68542.123.3 7.2 59.8 4.5 30.3 2.7 76.4 13.6 AD-68529.1 14.7 2.3 29.5 6.1 18.41.4 34.6 9.2 AD-68536.1 15.0 2.4 71.4 2.9 22.8 3.8 50.7 3.1 AD-68539.114.7 1.9 50.1 5.2 19.0 2.5 41.6 3.2 AD-68530.1 38.5 4.9 79.0 10.4 48.14.9 90.4 10.2 AD-68569.1 32.5 3.7 102.4 11.5 72.1 5.7 100.4 5.5AD-68564.1 13.4 5.5 44.0 3.7 84.8 12.1 94.6 4.2 AD-68547.1 99.5 18.289.5 8.8 37.5 3.9 85.4 5.4 AD-68548.1 100.3 10.3 89.5 2.2 58.2 7.7 93.55.0 AD-68544.1 69.5 8.7 98.0 2.3 20.8 3.5 60.8 7.8 AD-68557.1 114.6 14.393.9 5.8 48.2 8.9 93.9 0.7 AD-68535.1 80.0 7.3 87.0 14.9 30.1 3.7 88.63.4 AD-68567.1 34.3 14.6 73.6 39.9 66.3 18.3 94.2 4.6 AD-68538.1 32.93.8 80.1 12.8 30.0 3.2 71.8 3.9 AD-68532.1 37.8 11.7 70.8 6.2 24.5 1.559.5 9.0 AD-68528.1 50.5 7.0 81.6 8.4 28.9 1.9 73.2 6.8 AD-68553.1 104.712.4 108.3 12.8 29.3 4.8 52.9 6.6 AD-68554.1 103.5 16.7 107.4 7.7 33.64.6 86.9 3.5 AD-68545.1 130.1 23.3 112.8 9.1 45.9 2.2 97.8 9.3AD-68550.1 20.7 1.7 67.0 10.2 17.0 4.4 62.0 13.8 AD-68546.1 41.0 3.199.4 7.7 19.1 2.2 49.1 1.3 AD-68571.1 17.9 0.7 89.0 4.2 76.1 6.7 100.39.0 AD-68556.1 102.1 13.1 100.4 8.2 65.4 6.6 95.1 9.7 AD-68568.1 15.12.2 49.4 5.1 94.4 8.9 100.3 5.6 AD-68563.1 12.2 1.6 65.1 6.6 44.3 4.891.1 4.8 AD-68560.1 26.4 3.7 75.7 15.1 37.1 2.4 78.4 1.8 AD-68562.1 29.85.9 82.9 9.4 81.7 7.4 89.9 7.3 AD-68559.1 14.4 5.7 50.4 5.7 30.3 4.050.5 4.2 AD-68561.1 14.2 0.6 67.8 6.4 33.0 3.0 62.9 3.7 AD-68558.1 42.87.9 77.4 10.1 43.7 0.2 94.8 25.0 AD-68531.1 31.3 6.1 77.0 11.8 47.9 7.285.7 7.1 AD-68537.1 21.8 2.0 74.4 8.6 34.6 3.4 76.6 2.5 AD-68527.1 24.19.1 48.0 10.6 34.4 4.8 62.2 4.3 AD-68534.1 16.0 3.2 48.1 4.4 50.0 8.498.6 18.3 AD-68566.1 6.2 0.7 20.2 3.1 40.6 13.8 67.0 25.6 AD-1955 101.410.8 101.3 7.4 96.4 4.1 99.3 3.5

Example 2. Design, Synthesis, Selection, and In Vitro Evaluation ofAdditional iRNA Agents

This Example describes methods for the design, synthesis, selection, andin vitro evaluation of additional SCAP iRNA agents.

Bioinformatics

A set of additional siRNAs targeting human SCAP, “Homo sapiens SREBFchaperone” (human: NCBI refseqID NM_012235; NCBI GenelD: 22937) weredesigned using custom R and Python scripts. The human SCAP REFSEQ mRNAhas a length of 4268 bases.

A detailed list of the additional modified SCAP sense and antisensestrand sequences is shown in Table 5 and a detailed list of theadditional unmodified SCAP sense and antisense strand sequences is shownin Table 6.

In Vitro Hep3b Screening Cell Culture and Transfections

Hep3b cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM and 0.1μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat#13778-150) to 5 μl of siRNA duplexes per well into a 384-well plate andincubated at room temperature for 15 minutes. 40 μl of William's EMedium (Life Tech) containing ˜5×10³ cells were then added to the siRNAmixture. Cells were incubated for 24 hours prior to RNA purification.Single dose experiments were performed at 10 nM.

Isolation of total RNA, cDNA synthesis, and Real time PCR were performedusing the methods described for Example 1 above. The results from thissingle dose screen are shown in Table 7.

TABLE 5 SCAP Modified Sequences SEQ SEQ SEQ Duplex Sense Sequence IDAntisense Sequence ID mRNA target ID Name (5′ to 3′) NO (5′ to 3′) NOsequence NO AD-77471 UCAUCUGUCAGGCACUUUAdTdT  261UAAAGUGCCUGACAGAUGAdTdT  262 UCAUCUGUCAGGCACUUUG  263 AD-77472UUAAAAAACCAUAUCAUCAdTdT  264 UGAUGAUAUGGUUUUUUAAdTdT  265UUAAAAAACCAUAUCAUCA  266 AD-77473 AAUAAUAUUAAACUUUUUUdTdT  267AAAAAAGUUUAAUAUUAUUdTdT  268 AAUAAUAUUAAACUUUUUU  269 AD-77474CGCUGCCUCCUGACUGUAAdTdT  270 UUACAGUCAGGAGGCAGCGdTdT  271CGCUGCCUCCUGACUGUAA  272 AD-77475 AGUAUCUUCCAGCCGCUGAdTdT  273UCAGCGGCUGGAAGAUACUdTdT  274 AGUAUCUUCCAGCCGCUGC  275 AD-77476UUGGGGGAAAGAGCCGAGUdTdT  276 ACUCGGCUCUUUCCCCCAAdTdT  277UUGGGGGAAAGAGCCGAGU  278 AD-77477 CAAUGCACUGAACCUGGAAdTdT  279UUCCAGGUUCAGUGCAUUGdTdT  280 CAAUGCACUGAACCUGGAC  281 AD-77478GGGCCAAUGCACUGAACCUdTdT  282 AGGUUCAGUGCAUUGGCCCdTdT  283GGGCCAAUGCACUGAACCU  284 AD-77479 CUGGGGUGCUGUGUGGGGAdTdT  285UCCCCACACAGCACCCCAGdTdT  286 CUGGGGUGCUGUGUGGGGG  287 AD-77480UUGCCCAGGCAGGAGGCUAdTdT  288 UAGCCUCCUGCCUGGGCAAdTdT  289UUGCCCAGGCAGGAGGCUG  290 AD-77481 CCUCCUUGCCCAGGCAGGAdTdT  291UCCUGCCUGGGCAAGGAGGdTdT  292 CCUCCUUGCCCAGGCAGGA  293 AD-77482UGGACUGAGCGCAGGGCCUdTdT  294 AGGCCCUGCGCUCAGUCCAdTdT  295UGGACUGAGCGCAGGGCCU  296 AD-77483 CUCUGUGCUGGAGAAGCUAdTdT  297UAGCUUCUCCAGCACAGAGdTdT  298 CUCUGUGCUGGAGAAGCUG  299 AD-77484UGCCAUUGUCUGCAACUUUdTdT  300 AAAGUUGCAGACAAUGGCAdTdT  301UGCCAUUGUCUGCAACUUU  302 AD-77485 CUGGUGCUGGACAACGCUAdTdT  303UAGCGUUGUCCAGCACCAGdTdT  304 CUGGUGCUGGACAACGCUG  305 AD-77486GCCAGAUCCUGGUGCUGGAdTdT  306 UCCAGCACCAGGAUCUGGCdTdT  307GCCAGAUCCUGGUGCUGGA  308 AD-77487 GAGGCCCAGCCUGCCCGCAdTdT  309UGCGGGCAGGCUGGGCCUCdTdT  310 GAGGCCCAGCCUGCCCGCC  311 AD-77488CCUGGGGAAGAACAGUGAAdTdT  312 UUCACUGUUCUUCCCCAGGdTdT  313CCUGGGGAAGAACAGUGAG  314 AD-77489 UGUUACAGACAGUCUACCUdTdT  315AGGUAGACUGUCUGUAACAdTdT  316 UGUUACAGACAGUCUACCU  317 AD-77490GUGUCUCCUUUUGGGACCUdTdT  318 AGGUCCCAAAAGGAGACACdTdT  319GUGUCUCCUUUUGGGACCU  320 AD-77491 GACUGGCGGCCAGGGCUGUdTdT  321ACAGCCCUGGCCGCCAGUCdTdT  322 GACUGGCGGCCAGGGCUGU  323 AD-77492ACAACCUGCUGGUGACUGAdTdT  324 UCAGUCACCAGCAGGUUGUdTdT  325ACAACCUGCUGGUGACUGG  326 AD-77493 UUGGGUGUCAUCUCAGACAdTdT  327UGUCUGAGAUGACACCCAAdTdT  328 UUGGGUGUCAUCUCAGACA  329 AD-77494AAGCUUGGGUGUCAUCUCAdTdT  330 UGAGAUGACACCCAAGCUUdTdT  331AAGCUUGGGUGUCAUCUCA  332 AD-77495 ACCUGGGCUGUGGUGCAAAdTdT  333UUUGCACCACAGCCCAGGUdTdT  334 ACCUGGGCUGUGGUGCAAG  335 AD-77496GCACAGGCAUCAAGUUCUAdTdT  336 UAGAACUUGAUGCCUGUGCdTdT  337GCACAGGCAUCAAGUUCUA  338 AD-77497 CAGCAUCUGGGACCGCAGAdTdT  339UCUGCGGUCCCAGAUGCUGdTdT  340 CAGCAUCUGGGACCGCAGC  341 AD-77498GGCCUGGAUGACCUCAUCAdTdT  342 UGAUGAGGUCAUCCAGGCCdTdT  343GGCCUGGAUGACCUCAUCA  344 AD-77499 CCUGUGUCAUCAGCAGUGAdTdT  345UCACUGCUGAUGACACAGGdTdT  346 CCUGUGUCAUCAGCAGUGG  347 AD-77500UACCUGUACCACCUCCUGUdTdT  348 ACAGGAGGUGGUACAGGUAdTdT  349UACCUGUACCACCUCCUGU  350 AD-77501 GGGGAUGUCACCUCCCUUAdTdT  351UAAGGGAGGUGACAUCCCCdTdT  352 GGGGAUGUCACCUCCCUUA  353 AD-77502UUGCUCACCGUGGGGAUGUdTdT  354 ACAUCCCCACGGUGAGCAAdTdT  355UUGCUCACCGUGGGGAUGU  356 AD-77503 AGCCGGGUCAGCCAUGUGUdTdT  357ACACAUGGCUGACCCGGCUdTdT  358 AGCCGGGUCAGCCAUGUGU  359 AD-77504UCUGCCUGUGGGAUGUACUdTdT  360 AGUACAUCCCACAGGCAGAdTdT  361UCUGCCUGUGGGAUGUACU  362 AD-77505 AGGACAAGAUGGGGCCAUAdTdT  363UAUGGCCCCAUCUUGUCCUdTdT  364 AGGACAAGAUGGGGCCAUC  365 AD-77506AUGGUGCUGGCCAGUGGAAdTdT  366 UUCCACUGGCCAGCACCAUdTdT  367AUGGUGCUGGCCAGUGGAG  368 AD-77507 CAUCACGACCGUGUACAUUdTdT  369AAUGUACACGGUCGUGAUGdTdT  370 CAUCACGACCGUGUACAUU  371 AD-77508ACUCAGGGGCCAUCACGAAdTdT  372 UUCGUGAUGGCCCCUGAGUdTdT  373ACUCAGGGGCCAUCACGAC  374 AD-77509 UUCACCCUUCAGGGCCACUdTdT  375AGUGGCCCUGAAGGGUGAAdTdT  376 UUCACCCUUCAGGGCCACU  377 AD-77510AGGACUCGUGCUGCCUCUUdTdT  378 AAGAGGCAGCACGAGUCCUdTdT  379AGGACUCGUGCUGCCUCUU  380 AD-77511 UUCCGUCUGGAGGACUCGUdTdT  381ACGAGUCCUCCAGACGGAAdTdT  382 UUCCGUCUGGAGGACUCGU  383 AD-77512AGUGUUCCGUCUGGAGGAAdTdT  384 UUCCUCCAGACGGAACACUdTdT  385AGUGUUCCGUCUGGAGGAC  386 AD-77513 AAGACCACACACUGAGAGUdTdT  387ACUCUCAGUGUGUGGUCUUdTdT  388 AAGACCACACACUGAGAGU  389 AD-77514UUGGUGACUGGGAGCCAAAdTdT  390 UUUGGCUCCCAGUCACCAAdTdT  391UUGGUGACUGGGAGCCAAG  392 AD-77515 GGGCGCUUGGUGACUGGGAdTdT  393UCCCAGUCACCAAGCGCCCdTdT  394 GGGCGCUUGGUGACUGGGA  395 AD-77516CCUGAAAGCCGCUGCUGGAdTdT  396 UCCAGCAGCGGCUUUCAGGdTdT  397CCUGAAAGCCGCUGCUGGG  398 AD-77517 AAAAACCCAUCACAGCCCUdTdT  399AGGGCUGUGAUGGGUUUUUdTdT  400 AAAAACCCAUCACAGCCCU  401 AD-77518AGUGCCCUGUGCACACCAAdTdT  402 UUGGUGUGCACAGGGCACUdTdT  403AGUGCCCUGUGCACACCAA  404 AD-77519 GACCCACACAGUGCCCUGUdTdT  405ACAGGGCACUGUGUGGGUCdTdT  406 GACCCACACAGUGCCCUGU  407 AD-77520UACAGCAGCAGCGACACAAdTdT  408 UUGUGUCGCUGCUGCUGUAdTdT  409UACAGCAGCAGCGACACAG  410 AD-77521 CCCCUGCCUCUCCAGUGUAdTdT  411UACACUGGAGAGGCAGGGGdTdT  412 CCCCUGCCUCUCCAGUGUA  413 AD-77522GCGGGGCAGUUCCCCUGCAdTdT  414 UGCAGGGGAACUGCCCCGCdTdT  415GCGGGGCAGUUCCCCUGCC  416 AD-77523 UUAGAGGGACCCCAGGGCAdTdT  417UGCCCUGGGGUCCCUCUAAdTdT  418 UUAGAGGGACCCCAGGGCG  419 AD-77524CCCUCAGCCCCCUGCAGUUdTdT  420 AACUGCAGGGGGCUGAGGGdTdT  421CCCUCAGCCCCCUGCAGUU  422 AD-77525 UUGGAGACCCACACUGCCAdTdT  423UGGCAGUGUGGGUCUCCAAdTdT  424 UUGGAGACCCACACUGCCC  425 AD-77526CCCUUGAUUUCUUCUCCUUdTdT  426 AAGGAGAAGAAAUCAAGGGdTdT  427CCCUUGAUUUCUUCUCCUU  428 AD-77527 UGCACGGCUCAACGGUUCAdTdT  429UGAACCGUUGAGCCGUGCAdTdT  430 UGCACGGCUCAACGGUUCC  431 AD-77528UUCUUGGACAAAAGGAUUAdTdT  432 UAAUCCUUUUGUCCAAGAAdTdT  433UUCUUGGACAAAAGGAUUG  434 AD-77529 CGCUCUGGUGUUCUUGGAAdTdT  435UUCCAAGAACACCAGAGCGdTdT  436 CGCUCUGGUGUUCUUGGAC  437 AD-77530UCUCCUCAGGCAUUACCGAdTdT  438 UCGGUAAUGCCUGAGGAGAdTdT  439UCUCCUCAGGCAUUACCGC  440 AD-77531 UGCAGCAGCGAGGAGGUCUdTdT  441AGACCUCCUCGCUGCUGCAdTdT  442 UGCAGCAGCGAGGAGGUCU  443 AD-77532ACGCCAUUGAAGGGGUGCUdTdT  444 AGCACCCCUUCAAUGGCGUdTdT  445ACGCCAUUGAAGGGGUGCU  446 AD-77533 GCGGAGCAGCGGCCGGCUAdTdT  447UAGCCGGCCGCUGCUCCGCdTdT  448 GCGGAGCAGCGGCCGGCUG  449 AD-77534ACCUCAUCGUGGUGGGGCAdTdT  450 UGCCCCACCACGAUGAGGUdTdT  451ACCUCAUCGUGGUGGGGCG  452 AD-77535 UUGGAGCUGCAGGGCAACAdTdT  453UGUUGCCCUGCAGCUCCAAdTdT  454 UUGGAGCUGCAGGGCAACC  455 AD-77536AGGGUUCCAUCUGGAGCUUdTdT  456 AAGCUCCAGAUGGAACCCUdTdT  457AGGGUUCCAUCUGGAGCUU  458 AD-77537 CCCAGUGCCGAGGGUUCCAdTdT  459UGGAACCCUCGGCACUGGGdTdT  460 CCCAGUGCCGAGGGUUCCA  461 AD-77538UUCCCUCGCCUGGGCCCCAdTdT  462 UGGGGCCCAGGCGAGGGAAdTdT  463UUCCCUCGCCUGGGCCCCC  464 AD-77539 CCCGAGAAAGGCUCCCCUUdTdT  465AAGGGGAGCCUUUCUCGGGdTdT  466 CCCGAGAAAGGCUCCCCUU  467 AD-77540GCUCCCCCGAGAAAGGCUAdTdT  468 UAGCCUUUCUCGGGGGAGCdTdT  469GCUCCCCCGAGAAAGGCUC  470 AD-77541 CCUGAGGACGAGGGUGGCUdTdT  471AGCCACCCUCGUCCUCAGGdTdT  472 CCUGAGGACGAGGGUGGCU  473 AD-77542CGGUGCUGUCCCAGGCCCAdTdT  474 UGGGCCUGGGACAGCACCGdTdT  475CGGUGCUGUCCCAGGCCCC  476 AD-77543 CCUCGCCUGGGCCGGUGCUdTdT  477AGCACCGGCCCAGGCGAGGdTdT  478 CCUCGCCUGGGCCGGUGCU  479 AD-77544CCAGCCCUGCGCCCACCCUdTdT  480 AGGGUGGGCGCAGGGCUGGdTdT  481CCAGCCCUGCGCCCACCCU  482 AD-77545 GCUGGCGGCCGUCUGCACAdTdT  483UGUGCAGACGGCCGCCAGCdTdT  484 GCUGGCGGCCGUCUGCACA  485 AD-77546UGUACCAGGAGGAGGGGCUdTdT  486 AGCCCCUCCUCCUGGUACAdTdT  487UGUACCAGGAGGAGGGGCU  488 AD-77547 CUGCCUGGUGCAGCGGGUAdTdT  489UACCCGCUGCACCAGGCAGdTdT  490 CUGCCUGGUGCAGCGGGUG  491 AD-77548UCUCGGGACUCCCCAGGCUdTdT  492 AGCCUGGGGAGUCCCGAGAdTdT  493UCUCGGGACUCCCCAGGCU  494 AD-77549 UGUGGCCGCUCUCGGGACUdTdT  495AGUCCCGAGAGCGGCCACAdTdT  496 UGUGGCCGCUCUCGGGACU  497 AD-77550CCGGCACCGGGCGGUCUGUdTdT  498 ACAGACCGCCCGGUGCCGGdTdT  499CCGGCACCGGGCGGUCUGU  500 AD-77551 CAGCCCGAGCCCCGGCACAdTdT  501UGUGCCGGGGCUCGGGCUGdTdT  502 CAGCCCGAGCCCCGGCACC  503 AD-77552GUCCUCACAGCCCACUCAAdTdT  504 UUGAGUGGGCUGUGAGGACdTdT  505GUCCUCACAGCCCACUCAG  506 AD-77553 UUUCAGCGCAGCCUCGGUAdTdT  507UACCGAGGCUGCGCUGAAAdTdT  508 UUUCAGCGCAGCCUCGGUC  509 AD-77554UUAAUUGACACCAACUUUUdTdT  510 AAAAGUUGGUGUCAAUUAAdTdT  511UUAAUUGACACCAACUUUU  512 AD-77555 ACCUCACCUGCUUAAUUGAdTdT  513UCAAUUAAGCAGGUGAGGUdTdT  514 ACCUCACCUGCUUAAUUGA  515 AD-77556UUCGGGGACCAGCCUGACAdTdT  516 UGUCAGGCUGGUCCCCGAAdTdT  517UUCGGGGACCAGCCUGACC  518 AD-77557 CUCCGCCGCCUUCCCUCUUdTdT  519AAGAGGGAAGGCGGCGGAGdTdT  520 CUCCGCCGCCUUCCCUCUU  521 AD-77558CCUCCCCUGAGACACCGCAdTdT  522 UGCGGUGUCUCAGGGGAGGdTdT  523CCUCCCCUGAGACACCGCC  524 AD-77561 CCUAAGCAGCGAGAGCUGAdTdT  525UCAGCUCUCGCUGCUUAGGdTdT  526 CCUAAGCAGCGAGAGCUGG  527 AD-77562CUGCGGAUCGCCCAAGGCAdTdT  528 UGCCUUGGGCGAUCCGCAGdTdT  529CUGCGGAUCGCCCAAGGCC  530 AD-77563 CCUGGAGGUGAAGCUGCGAdTdT  531UCGCAGCUUCACCUCCAGGdTdT  532 CCUGGAGGUGAAGCUGCGG  533 AD-77564UCUCAACCCCGGUAGACCUdTdT  534 AGGUCUACCGGGGUUGAGAdTdT  535UCUCAACCCCGGUAGACCU  536 AD-77565 AAGUCUGUGGUCUCAACCAdTdT  537UGGUUGAGACCACAGACUUdTdT  538 AAGUCUGUGGUCUCAACCC  539 AD-77566UUAUUGGGUUAGAGAAUGUdTdT  540 ACAUUCUCUAACCCAAUAAdTdT  541UUAUUGGGUUAGAGAAUGU  542 AD-77567 GGUGGUUAUUGGGUUAGAAdTdT  543UUCUAACCCAAUAACCACCdTdT  544 GGUGGUUAUUGGGUUAGAG  545 AD-77568UUUUCCCCUACCUUGUGGUdTdT  546 ACCACAAGGUAGGGGAAAAdTdT  547UUUUCCCCUACCUUGUGGU  548 AD-77569 CCUCAAUGGCGGCGAGAUUdTdT  549AAUCUCGCCGCCAUUGAGGdTdT  550 CCUCAAUGGCGGCGAGAUU  551 AD-77570UUCGGCCUGACGCCCACCAdTdT  552 UGGUGGGCGUCAGGCCGAAdTdT  553UUCGGCCUGACGCCCACCC  554 AD-77571 UGCACACUCUUCGGCCUGAdTdT  555UCAGGCCGAAGAGUGUGCAdTdT  556 UGCACACUCUUCGGCCUGA  557 AD-77572CAUGUCUGUGGGACUCUGAdTdT  558 UCAGAGUCCCACAGACAUGdTdT  559CAUGUCUGUGGGACUCUGC  560 AD-77573 GCUGCUCAUGUCUGUGGGAdTdT  561UCCCACAGACAUGAGCAGCdTdT  562 GCUGCUCAUGUCUGUGGGA  563 AD-77574UCACAGUGCUCAGCUCGCUdTdT  564 AGCGAGCUGAGCACUGUGAdTdT  565UCACAGUGCUCAGCUCGCU  566 AD-77575 GGCCCUGGCUGCCGUGGUAdTdT  567UACCACGGCAGCCAGGGCCdTdT  568 GGCCCUGGCUGCCGUGGUC  569 AD-77576CAAGUGGGGGCUGGCCCUAdTdT  570 UAGGGCCAGCCCCCACUUGdTdT  571CAAGUGGGGGCUGGCCCUG  572 AD-77577 UCGACAUGGUCAAGUCCAAdTdT  573UUGGACUUGACCAUGUCGAdTdT  574 UCGACAUGGUCAAGUCCAA  575 AD-77578UUCUCCACGCGGAAGAUCAdTdT  576 UGAUCUUCCGCGUGGAGAAdTdT  577UUCUCCACGCGGAAGAUCG  578 AD-77579 UGUUUGCCUACAUCUACUUdTdT  579AAGUAGAUGUAGGCAAACAdTdT  580 UGUUUGCCUACAUCUACUU  581 AD-77580GACCACCUACAUCAUCUUAdTdT  582 UAAGAUGAUGUAGGUGGUCdTdT  583GACCACCUACAUCAUCUUG  584 AD-77581 CAUCCCCCUUGUGACCACAdTdT  585UGUGGUCACAAGGGGGAUGdTdT  586 CAUCCCCCUUGUGACCACC  587 AD-77582UUGGUGUCGCUGAGCUCAUdTdT  588 AUGAGCUCAGCGACACCAAdTdT  589UUGGUGUCGCUGAGCUCAU  590 AD-77583 GCACUUCAAGGAGGAGAUUdTdT  591AAUCUCCUCCUUGAAGUGCdTdT  592 GCACUUCAAGGAGGAGAUU  593 AD-77584GAGAGCCUGGUCCACGUGAdTdT  594 UCACGUGGACCAGGCUCUCdTdT  595GAGAGCCUGGUCCACGUGC  596 AD-77585 ACUGCAGCCUUCGGGCGGAdTdT  597UCCGCCCGAAGGCUGCAGUdTdT  598 ACUGCAGCCUUCGGGCGGA  599 AD-77586UUCUGCACCCCAGCCCCAAdTdT  600 UUGGGGCUGGGGUGCAGAAdTdT  601UUCUGCACCCCAGCCCCAA  602 AD-77587 GUGCCCGCCUGAUGCUUCUdTdT  603AGAAGCAUCAGGCGGGCACdTdT  604 GUGCCCGCCUGAUGCUUCU  605 AD-77588UUCCUGGGCAGCCUGCGUAdTdT  606 UACGCAGGCUGCCCAGGAAdTdT  607UUCCUGGGCAGCCUGCGUG  608 AD-77589 UGCCAAGUUCCUGGGCAGAdTdT  609UCUGCCCAGGAACUUGGCAdTdT  610 UGCCAAGUUCCUGGGCAGC  611 AD-77590UCUUCCAGCACUACCAUGAdTdT  612 UCAUGGUAGUGCUGGAAGAdTdT  613UCUUCCAGCACUACCAUGC  614 AD-77591 UACACCAUCACCCUGGUCUdTdT  615AGACCAGGGUGAUGGUGUAdTdT  616 UACACCAUCACCCUGGUCU  617 AD-77592GGAAGAGGAUGGUCUCCUAdTdT  618 UAGGAGACCAUCCUCUUCCdTdT  619GGAAGAGGAUGGUCUCCUA  620 AD-77593 UCUACACCAGGAAGAGGAUdTdT  621AUCCUCUUCCUGGUGUAGAdTdT  622 UCUACACCAGGAAGAGGAU  623 AD-77594UACAGCGGGGUGAGCCUCUdTdT  624 AGAGGCUCACCCCGCUGUAdTdT  625UACAGCGGGGUGAGCCUCU  626 AD-77595 ACUUGUUAUUUGGUGUUCAdTdT  627UGAACACCAAAUAACAAGUdTdT  628 ACUUGUUAUUUGGUGUUCC  629 AD-77596UCAGCCACACUCAAAGACUdTdT  630 AGUCUUUGAGUGUGGCUGAdTdT  631UCAGCCACACUCAAAGACU  632 AD-77597 UGCAGACUUCAGCCACACUdTdT  633AGUGUGGCUGAAGUCUGCAdTdT  634 UGCAGACUUCAGCCACACU  635 AD-77598CACGAGCCUAAAACCCUGAdTdT  636 UCAGGGUUUUAGGCUCGUGdTdT  637CACGAGCCUAAAACCCUGC  638 AD-77599 UCCACCAGCACGAGCCUAAdTdT  639UUAGGCUCGUGCUGGUGGAdTdT  640 UCCACCAGCACGAGCCUAA  641 AD-77600GACAUCAUUGGGACCAUCAdTdT  642 UGAUGGUCCCAAUGAUGUCdTdT  643GACAUCAUUGGGACCAUCC  644 AD-77601 CUUCCAUGCUGAUCCUGAAdTdT  645UUCAGGAUCAGCAUGGAAGdTdT  646 CUUCCAUGCUGAUCCUGAC  647 AD-77602AGAAUGACUGGGAACGCUUdTdT  648 AAGCGUUCCCAGUCAUUCUdTdT  649AGAAUGACUGGGAACGCUU  650 AD-77603 UCUGGCAGAAUGACUGGGAdTdT  651UCCCAGUCAUUCUGCCAGAdTdT  652 UCUGGCAGAAUGACUGGGA  653 AD-77604CUGUCCCCUGGGAACUUCUdTdT  654 AGAAGUUCCCAGGGGACAGdTdT  655CUGUCCCCUGGGAACUUCU  656 AD-77605 CCUGCUGCUGUCCCCUGGAdTdT  657UCCAGGGGACAGCAGCAGGdTdT  658 CCUGCUGCUGUCCCCUGGG  659 AD-77606UCCCUGAGCAUGGAUGCCUdTdT  660 AGGCAUCCAUGCUCAGGGAdTdT  661UCCCUGAGCAUGGAUGCCU  662 AD-77607 CCUACUCCCUGAGCAUGGAdTdT  663UCCAUGCUCAGGGAGUAGGdTdT  664 CCUACUCCCUGAGCAUGGA  665 AD-77608UUAGGAAGCUCAGGAACCUdTdT  666 AGGUUCCUGAGCUUCCUAAdTdT  667UUAGGAAGCUCAGGAACCU  668 AD-77609 CGACCUGCUGCCAGGCCUUdTdT  669AAGGCCUGGCAGCAGGUCGdTdT  670 CGACCUGCUGCCAGGCCUU  671 AD-77610UGUGUCUGCAAGUGACCGAdTdT  672 UCGGUCACUUGCAGACACAdTdT  673UGUGUCUGCAAGUGACCGA  674 AD-77611 AGGAGCUUGGAGGAGUUGUdTdT  675ACAACUCCUCCAAGCUCCUdTdT  676 AGGAGCUUGGAGGAGUUGU  677 AD-77612GAGACAGCUCUGGGAUCAAdTdT  678 UUGAUCCCAGAGCUGUCUCdTdT  679GAGACAGCUCUGGGAUCAG  680 AD-77613 CGUGCUGAGAGACAGCUCUdTdT  681AGAGCUGUCUCUCAGCACGdTdT  682 CGUGCUGAGAGACAGCUCU  683 AD-77614GAGGAGAUCCGGAACCACAdTdT  684 UGUGGUUCCGGAUCUCCUCdTdT  685GAGGAGAUCCGGAACCACG  686 AD-77615 GGGCAUUCCAACUGGUGGAdTdT  687UCCACCAGUUGGAAUGCCCdTdT  688 GGGCAUUCCAACUGGUGGA  689 AD-77616UCGUUCACCUUUGUCCCGAdTdT  690 UCGGGACAAAGGUGAACGAdTdT  691UCGUUCACCUUUGUCCCGG  692 AD-77617 GUAUUUCGUUCACCUUUGUdTdT  693ACAAAGGUGAACGAAAUACdTdT  694 GUAUUUCGUUCACCUUUGU  695 AD-77618CCUCCUGGCAGUAGAUGUAdTdT  696 UACAUCUACUGCCAGGAGGdTdT  697CCUCCUGGCAGUAGAUGUA  698 AD-77619 UUCCCUGGCACAAGAACCUdTdT  699AGGUUCUUGUGCCAGGGAAdTdT  700 UUCCCUGGCACAAGAACCU  701 AD-77620UGUGAAGUCCUCAGUGUUUdTdT  702 AAACACUGAGGACUUCACAdTdT  703UGUGAAGUCCUCAGUGUUU  704 AD-77621 UUAUGUCCAGCAGAUAUUUdTdT  705AAAUAUCUGCUGGACAUAAdTdT  706 UUAUGUCCAGCAGAUAUUU  707 AD-77622CCCGGUGGCUUAUGUCCAAdTdT  708 UUGGACAUAAGCCACCGGGdTdT  709CCCGGUGGCUUAUGUCCAG  710 AD-77623 AGUGGUAUGUGGGUGCCCAdTdT  711UGGGCACCCACAUACCACUdTdT  712 AGUGGUAUGUGGGUGCCCC  713 AD-77624CCUACUGAGCAGCCUGAGUdTdT  714 ACUCAGGCUGCUCAGUAGGdTdT  715CCUACUGAGCAGCCUGAGU  716 AD-77625 CCACCUGUGGACUCUGACAdTdT  717UGUCAGAGUCCACAGGUGGdTdT  718 CCACCUGUGGACUCUGACC  719 AD-77626UUACUCGCCCCCACCUGUAdTdT  720 UACAGGUGGGGGCGAGUAAdTdT  721UUACUCGCCCCCACCUGUG  722 AD-77627 CCUGUGAAGGAUUACUCGAdTdT  723UCGAGUAAUCCUUCACAGGdTdT  724 CCUGUGAAGGAUUACUCGC  725 AD-77628UGUGGAAUUCACCACCCCUdTdT  726 AGGGGUGGUGAAUUCCACAdTdT  727UGUGGAAUUCACCACCCCU  728 AD-77629 CCUUGCCAGGAACAGGACAdTdT  729UGUCCUGUUCCUGGCAAGGdTdT  730 CCUUGCCAGGAACAGGACC  731 AD-77630UACCCACUGCUGAAACUCAdTdT  732 UGAGUUUCAGCAGUGGGUAdTdT  733UACCCACUGCUGAAACUCC  734 AD-77631 GCUGCUACCCACUGCUGAAdTdT  735UUCAGCAGUGGGUAGCAGCdTdT  736 GCUGCUACCCACUGCUGAA  737 AD-77632UUCUGCAUCUUAGCCUGCUdTdT  738 AGCAGGCUAAGAUGCAGAAdTdT  739UUCUGCAUCUUAGCCUGCU  740 AD-77633 UCAUCCUCUUCACAGGGUUdTdT  741AACCCUGUGAAGAGGAUGAdTdT  742 UCAUCCUCUUCACAGGGUU  743 AD-77634AUCCUAUCCCAUCCCCAUAdTdT  744 UAUGGGGAUGGGAUAGGAUdTdT  745AUCCUAUCCCAUCCCCAUC  746 AD-77635 UCUGUGCAUCCUAUCCCAUdTdT  747AUGGGAUAGGAUGCACAGAdTdT  748 UCUGUGCAUCCUAUCCCAU  749 AD-77636UACAACCAUGGGCUCCUCUdTdT  750 AGAGGAGCCCAUGGUUGUAdTdT  751UACAACCAUGGGCUCCUCU  752 AD-77637 CCUGACUGAAAGGCUGCGUdTdT  753ACGCAGCCUUUCAGUCAGGdTdT  754 CCUGACUGAAAGGCUGCGU  755 AD-77638UUCGGCUGAGGAUGACCCUdTdT  756 AGGGUCAUCCUCAGCCGAAdTdT  757UUCGGCUGAGGAUGACCCU  758 AD-77639 UUCCGGGAUGGCAGCAAGAdTdT  759UCUUGCUGCCAUCCCGGAAdTdT  760 UUCCGGGAUGGCAGCAAGG  761 AD-77640GGUGAUCCAUGGUCACUUUdTdT  762 AAAGUGACCAUGGAUCACCdTdT  763GGUGAUCCAUGGUCACUUU  764 AD-77641 UGUCAAGUGUGUGCCAGGAdTdT  765UCCUGGCACACACUUGACAdTdT  766 UGUCAAGUGUGUGCCAGGG  767 AD-77642UUGUUCUUUGUCAGUGCUAdTdT  768 UAGCACUGACAAAGAACAAdTdT  769UUGUUCUUUGUCAGUGCUG  770 AD-77643 UACCUGCACAUGUUGUUCUdTdT  771AGAACAACAUGUGCAGGUAdTdT  772 UACCUGCACAUGUUGUUCU  773 AD-77644GCUGCCACCACAGGUACCUdTdT  774 AGGUACCUGUGGUGGCAGCdTdT  775GCUGCCACCACAGGUACCU  776 AD-77645 CCGCAGCUUGGGAGGUGCUdTdT  777AGCACCUCCCAAGCUGCGGdTdT  778 CCGCAGCUUGGGAGGUGCU  779 AD-77646GCCGCCGCCGCCGCCGCAAdTdT  780 UUGCGGCGGCGGCGGCGGCdTdT  781GCCGCCGCCGCCGCCGCAG  782 AD-77647 UGCCGCCCCCGUCGCCGCAdTdT  783UGCGGCGACGGGGGCGGCAdTdT  784 UGCCGCCCCCGUCGCCGCC  785 AD-77648CCGCGCUCCGCCCCUGCUAdTdT  786 UAGCAGGGGCGGAGCGCGGdTdT  787CCGCGCUCCGCCCCUGCUG  788 AD-77649 GCACGCCGCGCUCCGCCCAdTdT  789UGGGCGGAGCGCGGCGUGCdTdT  790 GCACGCCGCGCUCCGCCCC  791 AD-77650GCGCCACGCACCGGACUGAdTdT  792 UCAGUCCGGUGCGUGGCGCdTdT  793GCGCCACGCACCGGACUGC  794 AD-77651 AGGAGAGAGAGGGAGGGCAdTdT  795UGCCCUCCCUCUCUCUCCUdTdT  796 AGGAGAGAGAGGGAGGGCG  797 AD-77652GCGGGCACCCGGCGGCCAAdTdT  798 UUGGCCGCCGGGUGCCCGCdTdT  799GCGGGCACCCGGCGGCCAG  800 AD-77653 UUCAGAUGGUGGGAAGGCUdTdT  801AGCCUUCCCACCAUCUGAAdTdT  802 UUCAGAUGGUGGGAAGGCU  803 AD-77654AGGAGAGCUGGGAACGACUdTdT  804 AGUCGUUCCCAGCUCUCCUdTdT  805AGGAGAGCUGGGAACGACU  806 AD-77655 CAGCGGGCUUGAGGCUCAAdTdT  807UUGAGCCUCAAGCCCGCUGdTdT  808 CAGCGGGCUUGAGGCUCAG  809 AD-77656CGGGACAGUGGCGUGGGCAdTdT  810 UGCCCACGCCACUGUCCCGdTdT  811CGGGACAGUGGCGUGGGCA  812 AD-77657 AGCGCCGGGACAGUGGCGUdTdT  813ACGCCACUGUCCCGGCGCUdTdT  814 AGCGCCGGGACAGUGGCGU  815 AD-77658CCGCGCCCAGGCAGGCAGAdTdT  816 UCUGCCUGCCUGGGCGCGGdTdT  817CCGCGCCCAGGCAGGCAGC  818 AD-77659 UUGCCUAACGCGCAUUCCAdTdT  819UGGAAUGCGCGUUAGGCAAdTdT  820 UUGCCUAACGCGCAUUCCG  821 AD-77660GACGCGCAGACCGGGGAUUdTdT  822 AAUCCCCGGUCUGCGCGUCdTdT  823GACGCGCAGACCGGGGAUU  824 AD-77661 UCUGCGUGUGGGACGCGCAdTdT  825UGCGCGUCCCACACGCAGAdTdT  826 UCUGCGUGUGGGACGCGCA  827 AD-77662GCAUGCUGCUGGUGAGCUAdTdT  828 UAGCUCACCAGCAGCAUGCdTdT  829GCAUGCUGCUGGUGAGCUG  830 AD-77663 UGCCUGGCCAGCGACGGCAdTdT  831UGCCGUCGCUGGCCAGGCAdTdT  832 UGCCUGGCCAGCGACGGCA  833 AD-77664CCUCAUGGACAUCGAGUGAdTdT  834 UCACUCGAUGUCCAUGAGGdTdT  835CCUCAUGGACAUCGAGUGC  836 AD-77665 UUGUGCUGCGCGGCCACCUdTdT  837AGGUGGCCGCGCAGCACAAdTdT  838 UUGUGCUGCGCGGCCACCU  839 AD-77666GACGGAGAUCGUGCCGCUUdTdT  840 AAGCGGCACGAUCUCCGUCdTdT  841GACGGAGAUCGUGCCGCUU  842 AD-77667 CCGAGACGGAGAUCGUGCAdTdT  843UGCACGAUCUCCGUCUCGGdTdT  844 CCGAGACGGAGAUCGUGCC  845 AD-77668UACGGCUAUGCGCCACCCAdTdT  846 UGGGUGGCGCAUAGCCGUAdTdT  847UACGGCUAUGCGCCACCCG  848 AD-77669 UGCCCUGCGACGACUACGAdTdT  849UCGUAGUCGUCGCAGGGCAdTdT  850 UGCCCUGCGACGACUACGG  851 AD-77670CGGAGGCGCGGGGAGCUGAdTdT  852 UCAGCUCCCCGCGCCUCCGdTdT  853CGGAGGCGCGGGGAGCUGC  854 AD-77671 CGCAACUACGGGCAGCUGAdTdT  855UCAGCUGCCCGUAGUUGCGdTdT  856 CGCAACUACGGGCAGCUGG  857 AD-77672GCUAUGCCCGCGCAACUAAdTdT  858 UUAGUUGCGCGGGCAUAGCdTdT  859GCUAUGCCCGCGCAACUAC  860 AD-77673 UCUGCCUCUACCGCGUGCUdTdT  861AGCACGCGGUAGAGGCAGAdTdT  862 UCUGCCUCUACCGCGUGCU  863 AD-77674UGGCCACCGGCAUCGUCUUdTdT  864 AAGACGAUGCCGGUGGCCAdTdT  865UGGCCACCGGCAUCGUCUU  866 AD-77675 GCUGGGCCUGGCCACCGGAdTdT  867UCCGGUGGCCAGGCCCAGCdTdT  868 GCUGGGCCUGGCCACCGGC  869 AD-77676UGUACAAGGUGGCGGCGCUdTdT  870 AGCGCCGCCACCUUGUACAdTdT  871UGUACAAGGUGGCGGCGCU  872 AD-77677 UCACGCUGUACAAGGUGGAdTdT  873UCCACCUUGUACAGCGUGAdTdT  874 UCACGCUGUACAAGGUGGC  875 AD-77678CAGGCCCAUGGAGACGUCAdTdT  876 UGACGUCUCCAUGGGCCUGdTdT  877CAGGCCCAUGGAGACGUCA  878 AD-77679 AGGUGGGGUGCAGGCCCAUdTdT  879AUGGGCCUGCACCCCACCUdTdT  880 AGGUGGGGUGCAGGCCCAU  881 AD-77680CAGGACCCAAGGGCCCAGAdTdT  882 UCUGGGCCCUUGGGUCCUGdTdT  883CAGGACCCAAGGGCCCAGG  884 AD-77681 UGCUGGGCACUGGGAAGCAdTdT  885UGCUUCCCAGUGCCCAGCAdTdT  886 UGCUGGGCACUGGGAAGCA  887 AD-77682CCACCGGGGCCCAUACCUAdTdT  888 UAGGUAUGGGCCCCGGUGGdTdT  889CCACCGGGGCCCAUACCUG  890 AD-77683 ACCCUCAGGACGGCCGCAAdTdT  891UUGCGGCCGUCCUGAGGGUdTdT  892 ACCCUCAGGACGGCCGCAG  893 AD-77684GCUCUGGAGGGCCGGCACAdTdT  894 UGUGCCGGCCCUCCAGAGCdTdT  895GCUCUGGAGGGCCGGCACC  896 AD-77685 GCUCCGCCUGAACCCGAGAdTdT  897UCUCGGGUUCAGGCGGAGCdTdT  898 GCUCCGCCUGAACCCGAGG  899 AD-77686CCGUCAUCCCAGUCACGCUdTdT  900 AGCGUGACUGGGAUGACGGdTdT  901CCGUCAUCCCAGUCACGCU  902 AD-77687 UACAUCAGCCUGCUGCCCAdTdT  903UGGGCAGCAGGCUGAUGUAdTdT  904 UACAUCAGCCUGCUGCCCG  905 AD-77688CCAAGAGGUACAUCAGCCUdTdT  906 AGGCUGAUGUACCUCUUGGdTdT  907CCAAGAGGUACAUCAGCCU  908 AD-77689 UACAACAUCACACUGGCCAdTdT  909UGGCCAGUGUGAUGUUGUAdTdT  910 UACAACAUCACACUGGCCA  911 AD-77690CGACGCUCUUCAGCUAUUAdTdT  912 UAAUAGCUGAAGAGCGUCGdTdT  913CGACGCUCUUCAGCUAUUA  914 AD-77691 UCCGCCACUGGCCGACGCUdTdT  915AGCGUCGGCCAGUGGCGGAdTdT  916 UCCGCCACUGGCCGACGCU  917 AD-77692AUUGUCCUUCCGCCACUGAdTdT  918 UCAGUGGCGGAAGGACAAUdTdT  919AUUGUCCUUCCGCCACUGG  920 AD-77693 AGGAACUUUGGAGGAAAUUdTdT  921AAUUUCCUCCAAAGUUCCUdTdT  922 AGGAACUUUGGAGGAAAUU  923 AD-77694CUGGGGGCCUGAGGAUGAAdTdT  924 UUCAUCCUCAGGCCCCCAGdTdT  925CUGGGGGCCUGAGGAUGAG  926 AD-77695 AGUCCCAGAGGUAACCUGAdTdT  927UCAGGUUACCUCUGGGACUdTdT  928 AGUCCCAGAGGUAACCUGG  929 AD-77696UUGUCCAUGACAGCCCAGUdTdT  930 ACUGGGCUGUCAUGGACAAdTdT  931UUGUCCAUGACAGCCCAGU  932 AD-77697 AGGUCCAGCAGAGGUUGUAdTdT  933UACAACCUCUGCUGGACCUdTdT  934 AGGUCCAGCAGAGGUUGUC  935 AD-77698AGUCACCUGAGCGUGGAGAdTdT  936 UCUCCACGCUCAGGUGACUdTdT  937AGUCACCUGAGCGUGGAGG  938 AD-77699 CAGACGUCGCCAGGCGAGUdTdT  939ACUCGCCUGGCGACGUCUGdTdT  940 CAGACGUCGCCAGGCGAGU  941 AD-77700UAAGCUACCUGAGAACCAAdTdT  942 UUGGUUCUCAGGUAGCUUAdTdT  943UAAGCUACCUGAGAACCAG  944 AD-77701 CUGAUGCCCCUAAGCUACAdTdT  945UGUAGCUUAGGGGCAUCAGdTdT  946 CUGAUGCCCCUAAGCUACC  947 AD-77702UUCUCCAUCUUCCCACCUAdTdT  948 UAGGUGGGAAGAUGGAGAAdTdT  949UUCUCCAUCUUCCCACCUG  950 AD-77703 UGCCUUCUCCAUCUUCCCAdTdT  951UGGGAAGAUGGAGAAGGCAdTdT  952 UGCCUUCUCCAUCUUCCCA  953 AD-77704CCAGCCACCCGGACCCUGAdTdT  954 UCAGGGUCCGGGUGGCUGGdTdT  955CCAGCCACCCGGACCCUGC  956 AD-77705 UGCCCCCCAGCCACCCGGAdTdT  957UCCGGGUGGCUGGGGGGCAdTdT  958 UGCCCCCCAGCCACCCGGA  959 AD-77706GUGCCUAGUGGCAUGCUGAdTdT  960 UCAGCAUGCCACUAGGCACdTdT  961GUGCCUAGUGGCAUGCUGC  962 AD-77707 CCUGGCUCCCAUGCCCGUAdTdT  963UACGGGCAUGGGAGCCAGGdTdT  964 CCUGGCUCCCAUGCCCGUG  965 AD-77708CAUUGGGUGAGGGAGCCCUdTdT  966 AGGGCUCCCUCACCCAAUGdTdT  967CAUUGGGUGAGGGAGCCCU  968 AD-77709 CAGGUGACGGAACAGAGCAdTdT  969UGCUCUGUUCCGUCACCUGdTdT  970 CAGGUGACGGAACAGAGCC  971 AD-77710GCUGCCCAGGUGACGGAAAdTdT  972 UUUCCGUCACCUGGGCAGCdTdT  973GCUGCCCAGGUGACGGAAC  974 AD-77711 GCUGCGCAACUACCUCGCUdTdT  975AGCGAGGUAGUUGCGCAGCdTdT  976 GCUGCGCAACUACCUCGCU  977 AD-77712ACACAGACCCAGCAGGGCUdTdT  978 AGCCCUGCUGGGUCUGUGUdTdT  979ACACAGACCCAGCAGGGCU  980 AD-77713 CCUGGUAUACACAGACCCAdTdT  981UGGGUCUGUGUAUACCAGGdTdT  982 CCUGGUAUACACAGACCCA  983 AD-77714UUGUCUGGAUUGGCAUCCUdTdT  984 AGGAUGCCAAUCCAGACAAdTdT  985UUGUCUGGAUUGGCAUCCU  986 AD-77715 CAUCAUGGCUGGCACCGUUdTdT  987AACGGUGCCAGCCAUGAUGdTdT  988 CAUCAUGGCUGGCACCGUU  989 AD-77716GCCUGGCACAGCGCCUCAUdTdT  990 AUGAGGCGCUGUGCCAGGCdTdT  991GCCUGGCACAGCGCCUCAU  992 AD-77717 UUCCUGGCCCGCACCCGCAdTdT  993UGCGGGUGCGGGCCAGGAAdTdT  994 UUCCUGGCCCGCACCCGCC  995 AD-77718GGCUGCGUGUUGUCUACUUdTdT  996 AAGUAGACAACACGCAGCCdTdT  997GGCUGCGUGUUGUCUACUU  998 AD-77719 UCCCCAAGAGGCUGCGUGUdTdT  999ACACGCAGCCUCUUGGGGAdTdT 1000 UCCCCAAGAGGCUGCGUGU 1001 AD-77720UUCCGAAACCUGCGGCUCAdTdT 1002 UGAGCCGCAGGUUUCGGAAdTdT 1003UUCCGAAACCUGCGGCUCC 1004 AD-77721 CGUUGCAGCCGUCUUCCUUdTdT 1005AAGGAAGACGGCUGCAACGdTdT 1006 CGUUGCAGCCGUCUUCCUU 1007 AD-77722CCCCACACCAUCACGUUGAdTdT 1008 UCAACGUGAUGGUGUGGGGdTdT 1009CCCCACACCAUCACGUUGC 1010 AD-77723 UGUGAGGCCGUCCACACCAdTdT 1011UGGUGUGGACGGCCUCACAdTdT 1012 UGUGAGGCCGUCCACACCC 1013 AD-77724GGCAGCUGGCUGUGAGGCAdTdT 1014 UGCCUCACAGCCAGCUGCCdTdT 1015GGCAGCUGGCUGUGAGGCC 1016 AD-77725 CCAACGCGCUACGAGCGGAdTdT 1017UCCGCUCGUAGCGCGUUGGdTdT 1018 CCAACGCGCUACGAGCGGC 1019 AD-77726CAAGCCAGUGGGACAGCCAdTdT 1020 UGGCUGUCCCACUGGCUUGdTdT 1021CAAGCCAGUGGGACAGCCA 1022 AD-77727 GCCUGCCUGCCCUCAGCCAdTdT 1023UGGCUGAGGGCAGGCAGGCdTdT 1024 GCCUGCCUGCCCUCAGCCA 1025 AD-77728GCGACUGCCCCCUGAGGCAdTdT 1026 UGCCUCAGGGGGCAGUCGCdTdT 1027GCGACUGCCCCCUGAGGCC 1028 AD-77729 UAGCAGACCUGAACAAGCAdTdT 1029UGCUUGUUCAGGUCUGCUAdTdT 1030 UAGCAGACCUGAACAAGCG 1031 AD-77730CAUUCGCCGGAUGGAGCUAdTdT 1032 UAGCUCCAUCCGGCGAAUGdTdT 1033CAUUCGCCGGAUGGAGCUA 1034 AD-77731 ACUGUCCUGUCCAUUGACAdTdT 1035UGUCAAUGGACAGGACAGUdTdT 1036 ACUGUCCUGUCCAUUGACA 1037 AD-77732UUCACCACUGUCCUGUCCAdTdT 1038 UGGACAGGACAGUGGUGAAdTdT 1039UUCACCACUGUCCUGUCCA 1040 AD-77733 UGUUUUUCACCACUGUCCUdTdT 1041AGGACAGUGGUGAAAAACAdTdT 1042 UGUUUUUCACCACUGUCCU 1043 AD-77734UUCUUCCUUCAGAUGCUGUdTdT 1044 ACAGCAUCUGAAGGAAGAAdTdT 1045UUCUUCCUUCAGAUGCUGU 1046 AD-77735 UGGUGUCUGACUUCUUCCUdTdT 1047AGGAAGAAGUCAGACACCAdTdT 1048 UGGUGUCUGACUUCUUCCU 1049 AD-77736UUUGCUGUCGUGGGGCUGAdTdT 1050 UCAGCCCCACGACAGCAAAdTdT 1051UUUGCUGUCGUGGGGCUGG 1052 AD-77737 UCUCUUUGCUGUCGUGGGAdTdT 1053UCCCACGACAGCAAAGAGAdTdT 1054 UCUCUUUGCUGUCGUGGGG 1055 AD-77738CCAUCCAGGAGUUCUGUCUdTdT 1056 AGACAGAACUCCUGGAUGGdTdT 1057CCAUCCAGGAGUUCUGUCU 1058 AD-77739 UUCACCCUAGUGCCCGCCAdTdT 1059UGGCGGGCACUAGGGUGAAdTdT 1060 UUCACCCUAGUGCCCGCCA 1061 AD-77740UCAUCCUCAUCGGCUACUUdTdT 1062 AAGUAGCCGAUGAGGAUGAdTdT 1063UCAUCCUCAUCGGCUACUU 1064 AD-77741 GCCACGGAGCUGGGCAUCAdTdT 1065UGAUGCCCAGCUCCGUGGCdTdT 1066 GCCACGGAGCUGGGCAUCA 1067 AD-77742CAUCAUGAAGAACAUGGCAdTdT 1068 UGCCAUGUUCUUCAUGAUGdTdT 1069CAUCAUGAAGAACAUGGCC 1070

TABLE 6 SCAP Unmodified Sequences SEQ SEQ Duplex Sense Sequence IDAntisense Sequence ID Position in Name (5′ to 3′) NO (5′ to 3′) NONM_012235.3 AD-77652 GCGGGCACCCGGCGGCCAA 1071 UUGGCCGCCGGGUGCCCGC 107217-35 AD-77651 AGGAGAGAGAGGGAGGGCA 1073 UGCCCUCCCUCUCUCUCCU 1074 34-52AD-77650 GCGCCACGCACCGGACUGA 1075 UCAGUCCGGUGCGUGGCGC 1076 50-68AD-77649 GCACGCCGCGCUCCGCCCA 1077 UGGGCGGAGCGCGGCGUGC 1078  82-100AD-77648 CCGCGCUCCGCCCCUGCUA 1079 UAGCAGGGGCGGAGCGCGG 1080  87-105AD-77647 UGCCGCCCCCGUCGCCGCA 1081 UGCGGCGACGGGGGCGGCA 1082 104-122AD-77646 GCCGCCGCCGCCGCCGCAA 1083 UUGCGGCGGCGGCGGCGGC 1084 120-138AD-77645 CCGCAGCUUGGGAGGUGCU 1085 AGCACCUCCCAAGCUGCGG 1086 133-151AD-77644 GCUGCCACCACAGGUACCU 1087 AGGUACCUGUGGUGGCAGC 1088 149-167AD-77643 UACCUGCACAUGUUGUUCU 1089 AGAACAACAUGUGCAGGUA 1090 163-181AD-77642 UUGUUCUUUGUCAGUGCUA 1091 UAGCACUGACAAAGAACAA 1092 175-193AD-77641 UGUCAAGUGUGUGCCAGGA 1093 UCCUGGCACACACUUGACA 1094 192-210AD-77640 GGUGAUCCAUGGUCACUUU 1095 AAAGUGACCAUGGAUCACC 1096 209-227AD-77639 UUCCGGGAUGGCAGCAAGA 1097 UCUUGCUGCCAUCCCGGAA 1098 226-244AD-77638 UUCGGCUGAGGAUGACCCU 1099 AGGGUCAUCCUCAGCCGAA 1100 249-267AD-77637 CCUGACUGAAAGGCUGCGU 1101 ACGCAGCCUUUCAGUCAGG 1102 265-283AD-77636 UACAACCAUGGGCUCCUCU 1103 AGAGGAGCCCAUGGUUGUA 1104 305-323AD-77635 UCUGUGCAUCCUAUCCCAU 1105 AUGGGAUAGGAUGCACAGA 1106 321-339AD-77634 AUCCUAUCCCAUCCCCAUA 1107 UAUGGGGAUGGGAUAGGAU 1108 328-346AD-77633 UCAUCCUCUUCACAGGGUU 1109 AACCCUGUGAAGAGGAUGA 1110 345-363AD-77632 UUCUGCAUCUUAGCCUGCU 1111 AGCAGGCUAAGAUGCAGAA 1112 362-380AD-77631 GCUGCUACCCACUGCUGAA 1113 UUCAGCAGUGGGUAGCAGC 1114 378-396AD-77630 UACCCACUGCUGAAACUCA 1115 UGAGUUUCAGCAGUGGGUA 1116 383-401AD-77629 CCUUGCCAGGAACAGGACA 1117 UGUCCUGUUCCUGGCAAGG 1118 402-420AD-77628 UGUGGAAUUCACCACCCCU 1119 AGGGGUGGUGAAUUCCACA 1120 421-439AD-77627 CCUGUGAAGGAUUACUCGA 1121 UCGAGUAAUCCUUCACAGG 1122 437-455AD-77626 UUACUCGCCCCCACCUGUA 1123 UACAGGUGGGGGCGAGUAA 1124 448-466AD-77625 CCACCUGUGGACUCUGACA 1125 UGUCAGAGUCCACAGGUGG 1126 458-476AD-77624 CCUACUGAGCAGCCUGAGU 1127 ACUCAGGCUGCUCAGUAGG 1128 491-509AD-77623 AGUGGUAUGUGGGUGCCCA 1129 UGGGCACCCACAUACCACU 1130 507-525AD-77622 CCCGGUGGCUUAUGUCCAA 1131 UUGGACAUAAGCCACCGGG 1132 523-541AD-77621 UUAUGUCCAGCAGAUAUUU 1133 AAAUAUCUGCUGGACAUAA 1134 532-550AD-77620 UGUGAAGUCCUCAGUGUUU 1135 AAACACUGAGGACUUCACA 1136 550-568AD-77619 UUCCCUGGCACAAGAACCU 1137 AGGUUCUUGUGCCAGGGAA 1138 567-585AD-77618 CCUCCUGGCAGUAGAUGUA 1139 UACAUCUACUGCCAGGAGG 1140 583-601AD-77617 GUAUUUCGUUCACCUUUGU 1141 ACAAAGGUGAACGAAAUAC 1142 599-617AD-77616 UCGUUCACCUUUGUCCCGA 1143 UCGGGACAAAGGUGAACGA 1144 604-622AD-77615 GGGCAUUCCAACUGGUGGA 1145 UCCACCAGUUGGAAUGCCC 1146 621-639AD-77614 GAGGAGAUCCGGAACCACA 1147 UGUGGUUCCGGAUCUCCUC 1148 638-656AD-77613 CGUGCUGAGAGACAGCUCU 1149 AGAGCUGUCUCUCAGCACG 1150 655-673AD-77612 GAGACAGCUCUGGGAUCAA 1151 UUGAUCCCAGAGCUGUCUC 1152 663-681AD-77611 AGGAGCUUGGAGGAGUUGU 1153 ACAACUCCUCCAAGCUCCU 1154 680-698AD-77610 UGUGUCUGCAAGUGACCGA 1155 UCGGUCACUUGCAGACACA 1156 696-714AD-77609 CGACCUGCUGCCAGGCCUU 1157 AAGGCCUGGCAGCAGGUCG 1158 712-730AD-77608 UUAGGAAGCUCAGGAACCU 1159 AGGUUCCUGAGCUUCCUAA 1160 729-747AD-77607 CCUACUCCCUGAGCAUGGA 1161 UCCAUGCUCAGGGAGUAGG 1162 745-763AD-77606 UCCCUGAGCAUGGAUGCCU 1163 AGGCAUCCAUGCUCAGGGA 1164 750-768AD-77605 CCUGCUGCUGUCCCCUGGA 1165 UCCAGGGGACAGCAGCAGG 1166 766-784AD-77604 CUGUCCCCUGGGAACUUCU 1167 AGAAGUUCCCAGGGGACAG 1168 773-791AD-77603 UCUGGCAGAAUGACUGGGA 1169 UCCCAGUCAUUCUGCCAGA 1170 789-807AD-77602 AGAAUGACUGGGAACGCUU 1171 AAGCGUUCCCAGUCAUUCU 1172 795-813AD-77601 CUUCCAUGCUGAUCCUGAA 1173 UUCAGGAUCAGCAUGGAAG 1174 811-829AD-77600 GACAUCAUUGGGACCAUCA 1175 UGAUGGUCCCAAUGAUGUC 1176 827-845AD-77599 UCCACCAGCACGAGCCUAA 1177 UUAGGCUCGUGCUGGUGGA 1178 843-861AD-77598 CACGAGCCUAAAACCCUGA 1179 UCAGGGUUUUAGGCUCGUG 1180 851-869AD-77597 UGCAGACUUCAGCCACACU 1181 AGUGUGGCUGAAGUCUGCA 1182 867-885AD-77596 UCAGCCACACUCAAAGACU 1183 AGUCUUUGAGUGUGGCUGA 1184 875-893AD-77595 ACUUGUUAUUUGGUGUUCA 1185 UGAACACCAAAUAACAAGU 1186 891-909AD-77594 UACAGCGGGGUGAGCCUCU 1187 AGAGGCUCACCCCGCUGUA 1188 917-935AD-77593 UCUACACCAGGAAGAGGAU 1189 AUCCUCUUCCUGGUGUAGA 1190 933-951AD-77592 GGAAGAGGAUGGUCUCCUA 1191 UAGGAGACCAUCCUCUUCC 1192 942-960AD-77591 UACACCAUCACCCUGGUCU 1193 AGACCAGGGUGAUGGUGUA 1194 959-977AD-77590 UCUUCCAGCACUACCAUGA 1195 UCAUGGUAGUGCUGGAAGA 1196 975-993AD-77589 UGCCAAGUUCCUGGGCAGA 1197 UCUGCCCAGGAACUUGGCA 1198  991-1009AD-77588 UUCCUGGGCAGCCUGCGUA 1199 UACGCAGGCUGCCCAGGAA 1200  998-1016AD-77587 GUGCCCGCCUGAUGCUUCU 1201 AGAAGCAUCAGGCGGGCAC 1202 1014-1032AD-77586 UUCUGCACCCCAGCCCCAA 1203 UUGGGGCUGGGGUGCAGAA 1204 1029-1047AD-77585 ACUGCAGCCUUCGGGCGGA 1205 UCCGCCCGAAGGCUGCAGU 1206 1047-1065AD-77584 GAGAGCCUGGUCCACGUGA 1207 UCACGUGGACCAGGCUCUC 1208 1064-1082AD-77583 GCACUUCAAGGAGGAGAUU 1209 AAUCUCCUCCUUGAAGUGC 1210 1081-1099AD-77582 UUGGUGUCGCUGAGCUCAU 1211 AUGAGCUCAGCGACACCAA 1212 1098-1116AD-77581 CAUCCCCCUUGUGACCACA 1213 UGUGGUCACAAGGGGGAUG 1214 1114-1132AD-77580 GACCACCUACAUCAUCUUA 1215 UAAGAUGAUGUAGGUGGUC 1216 1126-1144AD-77579 UGUUUGCCUACAUCUACUU 1217 AAGUAGAUGUAGGCAAACA 1218 1143-1161AD-77578 UUCUCCACGCGGAAGAUCA 1219 UGAUCUUCCGCGUGGAGAA 1220 1160-1178AD-77577 UCGACAUGGUCAAGUCCAA 1221 UUGGACUUGACCAUGUCGA 1222 1176-1194AD-77576 CAAGUGGGGGCUGGCCCUA 1223 UAGGGCCAGCCCCCACUUG 1224 1192-1210AD-77575 GGCCCUGGCUGCCGUGGUA 1225 UACCACGGCAGCCAGGGCC 1226 1204-1222AD-77574 UCACAGUGCUCAGCUCGCU 1227 AGCGAGCUGAGCACUGUGA 1228 1221-1239AD-77573 GCUGCUCAUGUCUGUGGGA 1229 UCCCACAGACAUGAGCAGC 1230 1237-1255AD-77572 CAUGUCUGUGGGACUCUGA 1231 UCAGAGUCCCACAGACAUG 1232 1243-1261AD-77571 UGCACACUCUUCGGCCUGA 1233 UCAGGCCGAAGAGUGUGCA 1234 1259-1277AD-77570 UUCGGCCUGACGCCCACCA 1235 UGGUGGGCGUCAGGCCGAA 1236 1268-1286AD-77569 CCUCAAUGGCGGCGAGAUU 1237 AAUCUCGCCGCCAUUGAGG 1238 1285-1303AD-77568 UUUUCCCCUACCUUGUGGU 1239 ACCACAAGGUAGGGGAAAA 1240 1302-1320AD-77567 GGUGGUUAUUGGGUUAGAA 1241 UUCUAACCCAAUAACCACC 1242 1318-1336AD-77566 UUAUUGGGUUAGAGAAUGU 1243 ACAUUCUCUAACCCAAUAA 1244 1323-1341AD-77565 AAGUCUGUGGUCUCAACCA 1245 UGGUUGAGACCACAGACUU 1246 1355-1373AD-77564 UCUCAACCCCGGUAGACCU 1247 AGGUCUACCGGGGUUGAGA 1248 1365-1383AD-77563 CCUGGAGGUGAAGCUGCGA 1249 UCGCAGCUUCACCUCCAGG 1250 1381-1399AD-77562 CUGCGGAUCGCCCAAGGCA 1251 UGCCUUGGGCGAUCCGCAG 1252 1394-1412AD-77561 CCUAAGCAGCGAGAGCUGA 1253 UCAGCUCUCGCUGCUUAGG 1254 1411-1429AD-77742 CAUCAUGAAGAACAUGGCA 1255 UGCCAUGUUCUUCAUGAUG 1256 1432-1450AD-77741 GCCACGGAGCUGGGCAUCA 1257 UGAUGCCCAGCUCCGUGGC 1258 1448-1466AD-77740 UCAUCCUCAUCGGCUACUU 1259 AAGUAGCCGAUGAGGAUGA 1260 1464-1482AD-77739 UUCACCCUAGUGCCCGCCA 1261 UGGCGGGCACUAGGGUGAA 1262 1481-1499AD-77738 CCAUCCAGGAGUUCUGUCU 1263 AGACAGAACUCCUGGAUGG 1264 1497-1515AD-77737 UCUCUUUGCUGUCGUGGGA 1265 UCCCACGACAGCAAAGAGA 1266 1513-1531AD-77736 UUUGCUGUCGUGGGGCUGA 1267 UCAGCCCCACGACAGCAAA 1268 1517-1535AD-77735 UGGUGUCUGACUUCUUCCU 1269 AGGAAGAAGUCAGACACCA 1270 1533-1551AD-77734 UUCUUCCUUCAGAUGCUGU 1271 ACAGCAUCUGAAGGAAGAA 1272 1544-1562AD-77733 UGUUUUUCACCACUGUCCU 1273 AGGACAGUGGUGAAAAACA 1274 1560-1578AD-77732 UUCACCACUGUCCUGUCCA 1275 UGGACAGGACAGUGGUGAA 1276 1565-1583AD-77731 ACUGUCCUGUCCAUUGACA 1277 UGUCAAUGGACAGGACAGU 1278 1571-1589AD-77730 CAUUCGCCGGAUGGAGCUA 1279 UAGCUCCAUCCGGCGAAUG 1280 1588-1606AD-77729 UAGCAGACCUGAACAAGCA 1281 UGCUUGUUCAGGUCUGCUA 1282 1605-1623AD-77728 GCGACUGCCCCCUGAGGCA 1283 UGCCUCAGGGGGCAGUCGC 1284 1621-1639AD-77727 GCCUGCCUGCCCUCAGCCA 1285 UGGCUGAGGGCAGGCAGGC 1286 1637-1655AD-77726 CAAGCCAGUGGGACAGCCA 1287 UGGCUGUCCCACUGGCUUG 1288 1654-1672AD-77725 CCAACGCGCUACGAGCGGA 1289 UCCGCUCGUAGCGCGUUGG 1290 1670-1688AD-77724 GGCAGCUGGCUGUGAGGCA 1291 UGCCUCACAGCCAGCUGCC 1292 1686-1704AD-77723 UGUGAGGCCGUCCACACCA 1293 UGGUGUGGACGGCCUCACA 1294 1696-1714AD-77722 CCCCACACCAUCACGUUGA 1295 UCAACGUGAUGGUGUGGGG 1296 1712-1730AD-77721 CGUUGCAGCCGUCUUCCUU 1297 AAGGAAGACGGCUGCAACG 1298 1725-1743AD-77720 UUCCGAAACCUGCGGCUCA 1299 UGAGCCGCAGGUUUCGGAA 1300 1742-1760AD-77719 UCCCCAAGAGGCUGCGUGU 1301 ACACGCAGCCUCUUGGGGA 1302 1758-1776AD-77718 GGCUGCGUGUUGUCUACUU 1303 AAGUAGACAACACGCAGCC 1304 1767-1785AD-77717 UUCCUGGCCCGCACCCGCA 1305 UGCGGGUGCGGGCCAGGAA 1306 1784-1802AD-77716 GCCUGGCACAGCGCCUCAU 1307 AUGAGGCGCUGUGCCAGGC 1308 1800-1818AD-77715 CAUCAUGGCUGGCACCGUU 1309 AACGGUGCCAGCCAUGAUG 1310 1816-1834AD-77714 UUGUCUGGAUUGGCAUCCU 1311 AGGAUGCCAAUCCAGACAA 1312 1833-1851AD-77713 CCUGGUAUACACAGACCCA 1313 UGGGUCUGUGUAUACCAGG 1314 1849-1867AD-77712 ACACAGACCCAGCAGGGCU 1315 AGCCCUGCUGGGUCUGUGU 1316 1857-1875AD-77711 GCUGCGCAACUACCUCGCU 1317 AGCGAGGUAGUUGCGCAGC 1318 1873-1891AD-77710 GCUGCCCAGGUGACGGAAA 1319 UUUCCGUCACCUGGGCAGC 1320 1889-1907AD-77709 CAGGUGACGGAACAGAGCA 1321 UGCUCUGUUCCGUCACCUG 1322 1895-1913AD-77708 CAUUGGGUGAGGGAGCCCU 1323 AGGGCUCCCUCACCCAAUG 1324 1914-1932AD-77707 CCUGGCUCCCAUGCCCGUA 1325 UACGGGCAUGGGAGCCAGG 1326 1930-1948AD-77706 GUGCCUAGUGGCAUGCUGA 1327 UCAGCAUGCCACUAGGCAC 1328 1946-1964AD-77705 UGCCCCCCAGCCACCCGGA 1329 UCCGGGUGGCUGGGGGGCA 1330 1962-1980AD-77704 CCAGCCACCCGGACCCUGA 1331 UCAGGGUCCGGGUGGCUGG 1332 1968-1986AD-77703 UGCCUUCUCCAUCUUCCCA 1333 UGGGAAGAUGGAGAAGGCA 1334 1984-2002AD-77702 UUCUCCAUCUUCCCACCUA 1335 UAGGUGGGAAGAUGGAGAA 1336 1988-2006AD-77701 CUGAUGCCCCUAAGCUACA 1337 UGUAGCUUAGGGGCAUCAG 1338 2004-2022AD-77700 UAAGCUACCUGAGAACCAA 1339 UUGGUUCUCAGGUAGCUUA 1340 2014-2032AD-77699 CAGACGUCGCCAGGCGAGU 1341 ACUCGCCUGGCGACGUCUG 1342 2030-2048AD-77698 AGUCACCUGAGCGUGGAGA 1343 UCUCCACGCUCAGGUGACU 1344 2046-2064AD-77697 AGGUCCAGCAGAGGUUGUA 1345 UACAACCUCUGCUGGACCU 1346 2062-2080AD-77696 UUGUCCAUGACAGCCCAGU 1347 ACUGGGCUGUCAUGGACAA 1348 2076-2094AD-77695 AGUCCCAGAGGUAACCUGA 1349 UCAGGUUACCUCUGGGACU 1350 2092-2110AD-77694 CUGGGGGCCUGAGGAUGAA 1351 UUCAUCCUCAGGCCCCCAG 1352 2107-2125AD-77693 AGGAACUUUGGAGGAAAUU 1353 AAUUUCCUCCAAAGUUCCU 1354 2124-2142AD-77692 AUUGUCCUUCCGCCACUGA 1355 UCAGUGGCGGAAGGACAAU 1356 2140-2158AD-77691 UCCGCCACUGGCCGACGCU 1357 AGCGUCGGCCAGUGGCGGA 1358 2148-2166AD-77690 CGACGCUCUUCAGCUAUUA 1359 UAAUAGCUGAAGAGCGUCG 1360 2160-2178AD-77689 UACAACAUCACACUGGCCA 1361 UGGCCAGUGUGAUGUUGUA 1362 2177-2195AD-77688 CCAAGAGGUACAUCAGCCU 1363 AGGCUGAUGUACCUCUUGG 1364 2193-2211AD-77687 UACAUCAGCCUGCUGCCCA 1365 UGGGCAGCAGGCUGAUGUA 1366 2201-2219AD-77686 CCGUCAUCCCAGUCACGCU 1367 AGCGUGACUGGGAUGACGG 1368 2217-2235AD-77685 GCUCCGCCUGAACCCGAGA 1369 UCUCGGGUUCAGGCGGAGC 1370 2233-2251AD-77684 GCUCUGGAGGGCCGGCACA 1371 UGUGCCGGCCCUCCAGAGC 1372 2255-2273AD-77683 ACCCUCAGGACGGCCGCAA 1373 UUGCGGCCGUCCUGAGGGU 1374 2271-2289AD-77682 CCACCGGGGCCCAUACCUA 1375 UAGGUAUGGGCCCCGGUGG 1376 2300-2318AD-77681 UGCUGGGCACUGGGAAGCA 1377 UGCUUCCCAGUGCCCAGCA 1378 2317-2335AD-77680 CAGGACCCAAGGGCCCAGA 1379 UCUGGGCCCUUGGGUCCUG 1380 2334-2352AD-77679 AGGUGGGGUGCAGGCCCAU 1381 AUGGGCCUGCACCCCACCU 1382 2350-2368AD-77678 CAGGCCCAUGGAGACGUCA 1383 UGACGUCUCCAUGGGCCUG 1384 2360-2378AD-77677 UCACGCUGUACAAGGUGGA 1385 UCCACCUUGUACAGCGUGA 1386 2376-2394AD-77676 UGUACAAGGUGGCGGCGCU 1387 AGCGCCGCCACCUUGUACA 1388 2382-2400AD-77675 GCUGGGCCUGGCCACCGGA 1389 UCCGGUGGCCAGGCCCAGC 1390 2398-2416AD-77674 UGGCCACCGGCAUCGUCUU 1391 AAGACGAUGCCGGUGGCCA 1392 2406-2424AD-77673 UCUGCCUCUACCGCGUGCU 1393 AGCACGCGGUAGAGGCAGA 1394 2439-2457AD-77672 GCUAUGCCCGCGCAACUAA 1395 UUAGUUGCGCGGGCAUAGC 1396 2455-2473AD-77671 CGCAACUACGGGCAGCUGA 1397 UCAGCUGCCCGUAGUUGCG 1398 2465-2483AD-77670 CGGAGGCGCGGGGAGCUGA 1399 UCAGCUCCCCGCGCCUCCG 1400 2501-2519AD-77669 UGCCCUGCGACGACUACGA 1401 UCGUAGUCGUCGCAGGGCA 1402 2517-2535AD-77668 UACGGCUAUGCGCCACCCA 1403 UGGGUGGCGCAUAGCCGUA 1404 2531-2549AD-77667 CCGAGACGGAGAUCGUGCA 1405 UGCACGAUCUCCGUCUCGG 1406 2547-2565AD-77666 GACGGAGAUCGUGCCGCUU 1407 AAGCGGCACGAUCUCCGUC 1408 2551-2569AD-77665 UUGUGCUGCGCGGCCACCU 1409 AGGUGGCCGCGCAGCACAA 1410 2568-2586AD-77664 CCUCAUGGACAUCGAGUGA 1411 UCACUCGAUGUCCAUGAGG 1412 2584-2602AD-77663 UGCCUGGCCAGCGACGGCA 1413 UGCCGUCGCUGGCCAGGCA 1414 2600-2618AD-77662 GCAUGCUGCUGGUGAGCUA 1415 UAGCUCACCAGCAGCAUGC 1416 2616-2634AD-77661 UCUGCGUGUGGGACGCGCA 1417 UGCGCGUCCCACACGCAGA 1418 2652-2670AD-77660 GACGCGCAGACCGGGGAUU 1419 AAUCCCCGGUCUGCGCGUC 1420 2663-2681AD-77659 UUGCCUAACGCGCAUUCCA 1421 UGGAAUGCGCGUUAGGCAA 1422 2680-2698AD-77658 CCGCGCCCAGGCAGGCAGA 1423 UCUGCCUGCCUGGGCGCGG 1424 2696-2714AD-77657 AGCGCCGGGACAGUGGCGU 1425 ACGCCACUGUCCCGGCGCU 1426 2712-2730AD-77656 CGGGACAGUGGCGUGGGCA 1427 UGCCCACGCCACUGUCCCG 1428 2717-2735AD-77655 CAGCGGGCUUGAGGCUCAA 1429 UUGAGCCUCAAGCCCGCUG 1430 2734-2752AD-77654 AGGAGAGCUGGGAACGACU 1431 AGUCGUUCCCAGCUCUCCU 1432 2751-2769AD-77653 UUCAGAUGGUGGGAAGGCU 1433 AGCCUUCCCACCAUCUGAA 1434 2770-2788AD-77558 CCUCCCCUGAGACACCGCA 1435 UGCGGUGUCUCAGGGGAGG 1436 2813-2831AD-77557 CUCCGCCGCCUUCCCUCUU 1437 AAGAGGGAAGGCGGCGGAG 1438 2841-2859AD-77556 UUCGGGGACCAGCCUGACA 1439 UGUCAGGCUGGUCCCCGAA 1440 2858-2876AD-77555 ACCUCACCUGCUUAAUUGA 1441 UCAAUUAAGCAGGUGAGGU 1442 2874-2892AD-77554 UUAAUUGACACCAACUUUU 1443 AAAAGUUGGUGUCAAUUAA 1444 2885-2903AD-77553 UUUCAGCGCAGCCUCGGUA 1445 UACCGAGGCUGCGCUGAAA 1446 2901-2919AD-77552 GUCCUCACAGCCCACUCAA 1447 UUGAGUGGGCUGUGAGGAC 1448 2917-2935AD-77551 CAGCCCGAGCCCCGGCACA 1449 UGUGCCGGGGCUCGGGCUG 1450 2933-2951AD-77550 CCGGCACCGGGCGGUCUGU 1451 ACAGACCGCCCGGUGCCGG 1452 2944-2962AD-77549 UGUGGCCGCUCUCGGGACU 1453 AGUCCCGAGAGCGGCCACA 1454 2960-2978AD-77548 UCUCGGGACUCCCCAGGCU 1455 AGCCUGGGGAGUCCCGAGA 1456 2969-2987AD-77547 CUGCCUGGUGCAGCGGGUA 1457 UACCCGCUGCACCAGGCAG 1458 2998-3016AD-77546 UGUACCAGGAGGAGGGGCU 1459 AGCCCCUCCUCCUGGUACA 1460 3015-3033AD-77545 GCUGGCGGCCGUCUGCACA 1461 UGUGCAGACGGCCGCCAGC 1462 3031-3049AD-77544 CCAGCCCUGCGCCCACCCU 1463 AGGGUGGGCGCAGGGCUGG 1464 3050-3068AD-77543 CCUCGCCUGGGCCGGUGCU 1465 AGCACCGGCCCAGGCGAGG 1466 3066-3084AD-77542 CGGUGCUGUCCCAGGCCCA 1467 UGGGCCUGGGACAGCACCG 1468 3078-3096AD-77541 CCUGAGGACGAGGGUGGCU 1469 AGCCACCCUCGUCCUCAGG 1470 3095-3113AD-77540 GCUCCCCCGAGAAAGGCUA 1471 UAGCCUUUCUCGGGGGAGC 1472 3111-3129AD-77539 CCCGAGAAAGGCUCCCCUU 1473 AAGGGGAGCCUUUCUCGGG 1474 3116-3134AD-77538 UUCCCUCGCCUGGGCCCCA 1475 UGGGGCCCAGGCGAGGGAA 1476 3133-3151AD-77537 CCCAGUGCCGAGGGUUCCA 1477 UGGAACCCUCGGCACUGGG 1478 3149-3167AD-77536 AGGGUUCCAUCUGGAGCUU 1479 AAGCUCCAGAUGGAACCCU 1480 3159-3177AD-77535 UUGGAGCUGCAGGGCAACA 1481 UGUUGCCCUGCAGCUCCAA 1482 3176-3194AD-77534 ACCUCAUCGUGGUGGGGCA 1483 UGCCCCACCACGAUGAGGU 1484 3192-3210AD-77533 GCGGAGCAGCGGCCGGCUA 1485 UAGCCGGCCGCUGCUCCGC 1486 3208-3226AD-77532 ACGCCAUUGAAGGGGUGCU 1487 AGCACCCCUUCAAUGGCGU 1488 3237-3255AD-77531 UGCAGCAGCGAGGAGGUCU 1489 AGACCUCCUCGCUGCUGCA 1490 3260-3278AD-77530 UCUCCUCAGGCAUUACCGA 1491 UCGGUAAUGCCUGAGGAGA 1492 3276-3294AD-77529 CGCUCUGGUGUUCUUGGAA 1493 UUCCAAGAACACCAGAGCG 1494 3292-3310AD-77528 UUCUUGGACAAAAGGAUUA 1495 UAAUCCUUUUGUCCAAGAA 1496 3302-3320AD-77527 UGCACGGCUCAACGGUUCA 1497 UGAACCGUUGAGCCGUGCA 1498 3325-3343AD-77526 CCCUUGAUUUCUUCUCCUU 1499 AAGGAGAAGAAAUCAAGGG 1500 3342-3360AD-77525 UUGGAGACCCACACUGCCA 1501 UGGCAGUGUGGGUCUCCAA 1502 3359-3377AD-77524 CCCUCAGCCCCCUGCAGUU 1503 AACUGCAGGGGGCUGAGGG 1504 3375-3393AD-77523 UUAGAGGGACCCCAGGGCA 1505 UGCCCUGGGGUCCCUCUAA 1506 3393-3411AD-77522 GCGGGGCAGUUCCCCUGCA 1507 UGCAGGGGAACUGCCCCGC 1508 3409-3427AD-77521 CCCCUGCCUCUCCAGUGUA 1509 UACACUGGAGAGGCAGGGG 1510 3420-3438AD-77520 UACAGCAGCAGCGACACAA 1511 UUGUGUCGCUGCUGCUGUA 1512 3437-3455AD-77519 GACCCACACAGUGCCCUGU 1513 ACAGGGCACUGUGUGGGUC 1514 3469-3487AD-77518 AGUGCCCUGUGCACACCAA 1515 UUGGUGUGCACAGGGCACU 1516 3478-3496AD-77517 AAAAACCCAUCACAGCCCU 1517 AGGGCUGUGAUGGGUUUUU 1518 3495-3513AD-77516 CCUGAAAGCCGCUGCUGGA 1519 UCCAGCAGCGGCUUUCAGG 1520 3511-3529AD-77515 GGGCGCUUGGUGACUGGGA 1521 UCCCAGUCACCAAGCGCCC 1522 3527-3545AD-77514 UUGGUGACUGGGAGCCAAA 1523 UUUGGCUCCCAGUCACCAA 1524 3533-3551AD-77513 AAGACCACACACUGAGAGU 1525 ACUCUCAGUGUGUGGUCUU 1526 3549-3567AD-77512 AGUGUUCCGUCUGGAGGAA 1527 UUCCUCCAGACGGAACACU 1528 3565-3583AD-77511 UUCCGUCUGGAGGACUCGU 1529 ACGAGUCCUCCAGACGGAA 1530 3569-3587AD-77510 AGGACUCGUGCUGCCUCUU 1531 AAGAGGCAGCACGAGUCCU 1532 3579-3597AD-77509 UUCACCCUUCAGGGCCACU 1533 AGUGGCCCUGAAGGGUGAA 1534 3596-3614AD-77508 ACUCAGGGGCCAUCACGAA 1535 UUCGUGAUGGCCCCUGAGU 1536 3612-3630AD-77507 CAUCACGACCGUGUACAUU 1537 AAUGUACACGGUCGUGAUG 1538 3622-3640AD-77506 AUGGUGCUGGCCAGUGGAA 1539 UUCCACUGGCCAGCACCAU 1540 3650-3668AD-77505 AGGACAAGAUGGGGCCAUA 1541 UAUGGCCCCAUCUUGUCCU 1542 3667-3685AD-77504 UCUGCCUGUGGGAUGUACU 1543 AGUACAUCCCACAGGCAGA 1544 3684-3702AD-77503 AGCCGGGUCAGCCAUGUGU 1545 ACACAUGGCUGACCCGGCU 1546 3710-3728AD-77502 UUGCUCACCGUGGGGAUGU 1547 ACAUCCCCACGGUGAGCAA 1548 3729-3747AD-77501 GGGGAUGUCACCUCCCUUA 1549 UAAGGGAGGUGACAUCCCC 1550 3740-3758AD-77500 UACCUGUACCACCUCCUGU 1551 ACAGGAGGUGGUACAGGUA 1552 3757-3775AD-77499 CCUGUGUCAUCAGCAGUGA 1553 UCACUGCUGAUGACACAGG 1554 3771-3789AD-77498 GGCCUGGAUGACCUCAUCA 1555 UGAUGAGGUCAUCCAGGCC 1556 3788-3806AD-77497 CAGCAUCUGGGACCGCAGA 1557 UCUGCGGUCCCAGAUGCUG 1558 3805-3823AD-77496 GCACAGGCAUCAAGUUCUA 1559 UAGAACUUGAUGCCUGUGC 1560 3822-3840AD-77495 ACCUGGGCUGUGGUGCAAA 1561 UUUGCACCACAGCCCAGGU 1562 3855-3873AD-77494 AAGCUUGGGUGUCAUCUCA 1563 UGAGAUGACACCCAAGCUU 1564 3871-3889AD-77493 UUGGGUGUCAUCUCAGACA 1565 UGUCUGAGAUGACACCCAA 1566 3875-3893AD-77492 ACAACCUGCUGGUGACUGA 1567 UCAGUCACCAGCAGGUUGU 1568 3891-3909AD-77491 GACUGGCGGCCAGGGCUGU 1569 ACAGCCCUGGCCGCCAGUC 1570 3904-3922AD-77490 GUGUCUCCUUUUGGGACCU 1571 AGGUCCCAAAAGGAGACAC 1572 3921-3939AD-77489 UGUUACAGACAGUCUACCU 1573 AGGUAGACUGUCUGUAACA 1574 3954-3972AD-77488 CCUGGGGAAGAACAGUGAA 1575 UUCACUGUUCUUCCCCAGG 1576 3970-3988AD-77487 GAGGCCCAGCCUGCCCGCA 1577 UGCGGGCAGGCUGGGCCUC 1578 3986-4004AD-77486 GCCAGAUCCUGGUGCUGGA 1579 UCCAGCACCAGGAUCUGGC 1580 4002-4020AD-77485 CUGGUGCUGGACAACGCUA 1581 UAGCGUUGUCCAGCACCAG 1582 4010-4028AD-77484 UGCCAUUGUCUGCAACUUU 1583 AAAGUUGCAGACAAUGGCA 1584 4027-4045AD-77483 CUCUGUGCUGGAGAAGCUA 1585 UAGCUUCUCCAGCACAGAG 1586 4075-4093AD-77482 UGGACUGAGCGCAGGGCCU 1587 AGGCCCUGCGCUCAGUCCA 1588 4092-4110AD-77481 CCUCCUUGCCCAGGCAGGA 1589 UCCUGCCUGGGCAAGGAGG 1590 4108-4126AD-77480 UUGCCCAGGCAGGAGGCUA 1591 UAGCCUCCUGCCUGGGCAA 1592 4113-4131AD-77479 CUGGGGUGCUGUGUGGGGA 1593 UCCCCACACAGCACCCCAG 1594 4129-4147AD-77478 GGGCCAAUGCACUGAACCU 1595 AGGUUCAGUGCAUUGGCCC 1596 4145-4163AD-77477 CAAUGCACUGAACCUGGAA 1597 UUCCAGGUUCAGUGCAUUG 1598 4149-4167AD-77476 UUGGGGGAAAGAGCCGAGU 1599 ACUCGGCUCUUUCCCCCAA 1600 4168-4186AD-77475 AGUAUCUUCCAGCCGCUGA 1601 UCAGCGGCUGGAAGAUACU 1602 4184-4202AD-77474 CGCUGCCUCCUGACUGUAA 1603 UUACAGUCAGGAGGCAGCG 1604 4197-4215AD-77473 AAUAAUAUUAAACUUUUUU 1605 AAAAAAGUUUAAUAUUAUU 1606 4214-4232AD-77472 UUAAAAAACCAUAUCAUCA 1607 UGAUGAUAUGGUUUUUUAA 1608 4231-4249AD-77471 UCAUCUGUCAGGCACUUUA 1609 UAAAGUGCCUGACAGAUGA 1610 4247-4265

TABLE 7 SCAP in vitro 10 nM screen Table 7 below presents the results ofa SCAP Single Dose (10 nM) Screen in Hep3b hepatocytes. Data areexpressed as percent message remaining relative to AD-1955 non-targetingcontrol. 10 nM Duplex Name AVG AD-77652 98.5 AD-77651 96.7 AD-77650 66.6AD-77649 80.9 AD-77648 83.9 AD-77647 76.0 AD-77646 81.1 AD-77645 73.3AD-77644 63.9 AD-77643 120.8 AD-77642 48.0 AD-77641 55.0 AD-77640 26.2AD-77639 95.3 AD-77638 72.9 AD-77637 74.5 AD-77636 79.8 AD-77635 43.0AD-77634 25.4 AD-77633 24.2 AD-77632 80.0 AD-77631 22.7 AD-77630 19.6AD-77629 23.5 AD-77628 61.6 AD-77627 14.9 AD-77626 39.2 AD-77625 18.6AD-77624 21.5 AD-77623 53.8 AD-77622 34.2 AD-77621 35.3 AD-77620 28.4AD-77619 98.1 AD-77618 31.9 AD-77617 25.9 AD-77616 50.6 AD-77615 50.9AD-77614 30.6 AD-77613 36.0 AD-77612 34.8 AD-77611 58.2 AD-77610 41.2AD-77609 71.1 AD-77608 130.3 AD-77607 33.0 AD-77606 77.5 AD-77605 33.2AD-77604 30.4 AD-77603 55.2 AD-77602 28.9 AD-77601 36.1 AD-77600 75.3AD-77599 31.3 AD-77598 32.8 AD-77597 59.9 AD-77596 31.7 AD-77595 26.8AD-77594 78.4 AD-77593 87.4 AD-77592 32.5 AD-77591 63.9 AD-77590 28.1AD-77589 45.3 AD-77588 52.0 AD-77587 19.8 AD-77586 29.5 AD-77585 61.3AD-77584 52.3 AD-77583 27.9 AD-77582 34.1 AD-77581 34.7 AD-77580 38.0AD-77579 28.4 AD-77578 28.1 AD-77577 42.0 AD-77576 55.6 AD-77575 28.8AD-77574 73.5 AD-77573 20.4 AD-77572 21.5 AD-77571 27.7 AD-77570 47.0AD-77569 28.2 AD-77568 67.4 AD-77567 19.1 AD-77566 59.2 AD-77565 51.1AD-77564 58.6 AD-77563 34.4 AD-77562 40.0 AD-77561 34.8 AD-77742 39.6AD-77741 42.1 AD-77740 32.0 AD-77739 116.0 AD-77738 20.9 AD-77737 36.0AD-77736 82.0 AD-77735 27.2 AD-77734 96.9 AD-77733 58.0 AD-77732 53.0AD-77731 19.0 AD-77730 21.9 AD-77729 20.6 AD-77728 39.5 AD-77727 76.9AD-77726 106.9 AD-77725 26.4 AD-77724 54.9 AD-77723 70.1 AD-77722 31.5AD-77721 16.7 AD-77720 29.4 AD-77719 48.2 AD-77718 16.6 AD-77717 99.1AD-77716 43.1 AD-77715 26.8 AD-77714 124.5 AD-77713 52.8 AD-77712 88.4AD-77711 48.9 AD-77710 33.4 AD-77709 32.1 AD-77708 125.4 AD-77707 123.5AD-77706 22.6 AD-77705 121.5 AD-77704 26.3 AD-77703 32.4 AD-77702 81.5AD-77701 19.6 AD-77700 27.0 AD-77699 51.6 AD-77698 36.2 AD-77697 36.8AD-77696 129.2 AD-77695 37.0 AD-77694 41.0 AD-77693 38.6 AD-77692 56.2AD-77691 102.5 AD-77690 18.7 AD-77689 68.7 AD-77688 21.5 AD-77687 50.2AD-77686 16.3 AD-77685 38.0 AD-77684 123.1 AD-77683 104.0 AD-77682 63.6AD-77681 91.8 AD-77680 86.1 AD-77679 51.4 AD-77678 39.2 AD-77677 70.0AD-77676 99.4 AD-77675 72.2 AD-77674 45.1 AD-77673 94.9 AD-77672 31.5AD-77671 47.3 AD-77670 62.9 AD-77669 22.5 AD-77668 73.7 AD-77667 22.5AD-77666 28.6 AD-77665 81.3 AD-77664 41.9 AD-77663 79.1 AD-77662 25.0AD-77661 79.2 AD-77660 24.7 AD-77659 82.0 AD-77658 74.0 AD-77657 73.5AD-77656 33.3 AD-77655 24.7 AD-77654 10.8 AD-77653 75.2 AD-77558 63.2AD-77557 50.5 AD-77556 57.6 AD-77555 9.7 AD-77554 27.2 AD-77553 60.1AD-77552 27.0 AD-77551 119.8 AD-77550 115.2 AD-77549 99.5 AD-77548 134.7AD-77547 23.4 AD-77546 108.5 AD-77545 38.4 AD-77544 147.7 AD-77543 123.0AD-77542 129.3 AD-77541 85.6 AD-77540 50.0 AD-77539 54.1 AD-77538 92.3AD-77537 59.2 AD-77536 108.2 AD-77535 102.2 AD-77534 32.2 AD-77533 44.2AD-77532 58.5 AD-77531 80.1 AD-77530 25.6 AD-77529 20.2 AD-77528 45.3AD-77527 26.3 AD-77526 25.2 AD-77525 59.2 AD-77524 34.8 AD-77523 87.9AD-77522 113.3 AD-77521 20.1 AD-77520 45.8 AD-77519 31.5 AD-77518 23.0AD-77517 57.8 AD-77516 49.8 AD-77515 35.0 AD-77514 22.2 AD-77513 25.3AD-77512 21.4 AD-77511 94.6 AD-77510 23.6 AD-77509 133.2 AD-77508 42.0AD-77507 20.8 AD-77506 43.3 AD-77505 20.6 AD-77504 83.3 AD-77503 32.5AD-77502 100.3 AD-77501 70.9 AD-77500 50.1 AD-77499 26.5 AD-77498 32.5AD-77497 45.9 AD-77496 28.9 AD-77495 35.5 AD-77494 24.4 AD-77493 54.3AD-77492 17.3 AD-77491 24.0 AD-77490 29.9 AD-77489 49.9 AD-77488 33.4AD-77487 66.5 AD-77486 18.1 AD-77485 32.0 AD-77484 35.5 AD-77483 18.6AD-77482 354.5 AD-77481 86.8 AD-77480 80.7 AD-77479 26.4 AD-77478 16.2AD-77477 16.2 AD-77476 28.1 AD-77475 11.1 AD-77474 14.6 AD-77473 76.3AD-77472 92.1 AD-77471 112.9 AD-1955 Avg 100.1

1. A double stranded ribonucleic acid (RNAi) agent for inhibitingexpression of a sterol regulatory element binding protein (SREBP)chaperone (SCAP) gene, wherein said dsRNA agent comprises a sense strandand an antisense strand, wherein said sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NOs:1-13; and wherein saidantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequencesof SEQ ID NOs:14-26.
 2. (canceled)
 3. The double stranded RNAi agent ofclaim 1, wherein the sense and antisense strands comprise sequencesselected from the group consisting of any one of the sequences in anyone of Tables 2, 3, 5 and
 6. 4. The double stranded RNAi agent of claim1, wherein the double stranded RNAi agent comprises at least onemodified nucleotide.
 5. The double stranded RNAi agent of claim 4,wherein at least one of the modified nucleotides is selected from thegroup consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT)nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anunlocked nucleotide, a conformationally restricted nucleotide, aconstrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modifiednucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modifiednucleotide, 2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modifiednucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, a nucleotide comprising a5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotidecomprising adenosine-glycol nucleic acid (GNA), a nucleotide comprisingthymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising2′-deoxythymidine-3′phosphate, a nucleotide comprising2′-deoxyguanosine-3′-phosphate, and a terminal nucleotide linked to acholesteryl derivative and a dodecanoic acid bisdecylamide group.
 6. Thedouble stranded RNAi agent of claim 4, further comprising at least onephosphorothioate internucleotide linkage.
 7. (canceled)
 8. The doublestranded RNAi agent of claim 1, wherein each strand is no more than 30nucleotides in length.
 9. The double stranded RNAi agent of claim 1,wherein at least one strand comprises a 3′ overhang of at least 1nucleotide; or a 3′ overhang of at least 2 nucleotides.
 10. The doublestranded RNAi agent of claim 1, wherein the double stranded RNAi agentfurther comprises an N-acetylgalactosamine (GalNAc) derivativeconjugated to the 3′ end of the sense strand through a monovalent orbranched bivalent or trivalent linker.
 11. The double stranded RNAiagent of claim 10, wherein the ligand is


12. The double stranded RNAi agent of claim 10, wherein the doublestranded RNAi agent is conjugated to the ligand as shown in thefollowing schematic

and, wherein X is O or S.
 13. (canceled)
 14. The double stranded RNAiagent of claim 10, wherein the ligand is a cholesterol.
 15. A doublestranded ribonucleic acid (RNAi) agent for inhibiting expression of asterol regulatory element binding protein (SREBP) chaperone (SCAP) gene,wherein said double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein said antisense strandcomprises a region complementary to part of an mRNA encoding SCAP,wherein each strand is about 14 to about 30 nucleotides in length,wherein said double stranded RNAi agent is represented by formula (III):sense:5′ n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q) 3′antisense:3′ n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′ (III)

wherein: j, k, and l are each independently 0 or 1; p, p′, q, and q′ areeach independently 0-6; each N_(a) and N_(a)′ independently representsan oligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides; each N_(b) and N_(b)′independently represents an oligonucleotide sequence comprising 0-10nucleotides which are either modified or unmodified or combinationsthereof; each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or maynot be present, independently represents an overhang nucleotide; XXX,YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent onemotif of three identical modifications on three consecutive nucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; and whereinthe sense strand is conjugated to at least one ligand.
 16. The doublestranded RNAi agent of claim 15, wherein formula (III) is represented byformula (IIIa): sense: 5′ n_(p)-N_(a)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIa);

wherein formula (III) is represented by formula (IIIb): sense:5′ n_(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′ 5′ (IIIb)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides; whereinformula (III) is represented by formula (IIIc): sense:5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n_(q)′ 5′ (IIIc)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides; or whereinformula (III) is represented by formula (IIId): sense:5′ n_(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)-Z Z Z-N_(a)-n_(q) 3′ antisense:3′ n_(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)′-n_(q)′ 5′(IIId)

wherein each N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 1-5 modified nucleotides and eachN_(a) and N_(a)′ independently represents an oligonucleotide sequencecomprising 2-10 modified nucleotides.
 17. (canceled)
 18. The doublestranded RNAi agent of claim 15, wherein the modifications on thenucleotides are selected from the group consisting of LNA, HNA, CeNA,2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro,2′-deoxy, 2′-hydroxyl, and combinations thereof.
 19. The double strandedRNAi agent of claim 15, wherein the ligand is one or more GalNAcderivatives attached through a bivalent or trivalent branched linker; ora cholesterol.
 20. (canceled)
 21. (canceled)
 22. A cell containing thedouble stranded RNAi agent of claim
 1. 23. A pharmaceutical compositionfor inhibiting expression of a SCAP gene comprising the double strandedRNAi agent of claim
 1. 24. A method of inhibiting for inhibitingexpression of a sterol regulatory element binding protein (SREBP)chaperone (SCAP) gene in a cell, the method comprising: (a) contactingthe cell with the double stranded RNAi agent of claim 1 or thepharmaceutical composition of claim 23; and (b) maintaining the cellproduced in step (a) for a time sufficient to obtain degradation of themRNA transcript of a SCAP gene, thereby inhibiting expression of theSCAP gene in the cell.
 25. The method of claim 24, wherein said cell iswithin a subject. 26.-29. (canceled)
 30. A method of treating a subjecthaving a disorder that would benefit from a reduction in SCAPexpression, comprising administering to the subject a therapeuticallyeffective amount of the double stranded RNAi agent of claim 1 or thepharmaceutical composition of claim 23, thereby treating said subject.31. The method of claim 30, wherein the subject suffers from aSCAP-associated disorder.
 32. The method of claim 30, wherein thesubject is a human.
 33. The method of claim 31, wherein theSCAP-associated disease is nonalcoholic fatty liver disease (NAFLD);fatty liver (steatosis); nonalcoholic steatohepatitis (NASH). 34.-40.(canceled)
 41. A kit for performing the method of claim 30, comprisinga) the double stranded RNAi agent, and b) instructions for use, and c)optionally, means for administering the double stranded RNAi agent tothe subject.